^k-vtic^.A^5~5  fH. 


Issued  April  7, 1911. 


U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  136. 
A.  D.  MELVIN,  Chief  of  Bureau. 


HE   DIAGNOSIS   OF   GLANDERS    BY 
COMPLEMENT  FIXATION. 


BY 


JOHN  R.  MOHLER,  V.  M.  D., 

Chief  of  the  Pathological  Division^ 

AND 

ADOLPH  EICHHORN,  D.  V.  S., 
Bacteriologist,  Pathological  Division. 


[Reprint,  October,  191  I,  with  sliglit  revision.] 


JAN  4    1943 
4-  i  B  R  A  R  y 


WASHINGTON: 

GOVERNMENT  PRINTING  OFFICE. 

1911. 


Digitized  by, the  Internet  Archive 

in  2007  With  funding  from 

IVIicrosoft  Corporatran 


http://www.archive.org/details/diagnosisofglandOOmohliala 


Issued  April  7, 1911. 

U.  S.  DEPARTMENT  OF  AGRICULTURE, 

BUREAU  OF  ANIMAL  INDUSTRY.— BULLETIN  136. 

A.  D.  MELVIN,  Chief  of  Bureau. 


THE   DIAGNOSIS   OF   GLANDERS    BY 
COMPLEMENT  FIXATION. 


BY 


JOHN  R.  MOHLER,  V.  M.  D., 

Chief  of  the  Pathological  Division, 
AND 

ADOLPH  EICHHORN,  D.  V.  S., 
Bacteriologist,  Pathological  Division. 


[Reprint,  October,  1911,  with  slight  revision.] 


WASHINGTON: 

GOVERNIVIENT  PRINTING  OFFICE. 

1911. 


THE  BUREAU  OF  ANIMAL  INDUSTRY. 


Chief:  A.  D.  Melvin. 

Assistant  Chief:  A.  M.  Fabeington. 

Chief  Clerk:  Chakles  C,  Carroll. 

Animal  Husbandry  Division:  George  M.  Rommel,  chief. 

Biochemic  Division:  M.  Dorset,  chief. 

Dairy  Division:  B.  H.  Rawl,  chief. 

Inspection  Division:  Rice  P.  Steddom,  chief;  R.  A.  Ramsay,  Morris  Wooden, 

and  Albert  E.  Behnke,  associate  chiefs. 
Pathological  Division:  John  R.  Mohler,  chief. 
Quarantine  Division:  Richard  W.  Hickman,  chief. 
Zoological  Division:  B.  H.  Ransom,  chief. 
Experiment  Station:  E.  C.  Schroeder,  superintendent. 
Editor:  James  M.  Pickens. 
2 


LETTER  OF  TRANSMITTAL. 


U.  S.  Department  of  Agriculture, 

Bureau  of  Animal  Industry, 

Washington,  D.  C,  March  W,  1911. 

Sir  :  I  have  the  honor  to  transmit  herewith  a  paper  entitled  "  The 
Diagnosis  of  Glanders  by  Complement  Fixation,"  by  Drs.  John  E. 
Mohler  and  Adoph  Eichhorn,  of  the  Pathological  Division  of  this 
bureau. 

Since  the  discovery  of  the  glanders  bacillus  in  1883  many  efforts 
have  been  made  to  find  a  reliable  method  of  making  an  early  diag- 
nosis of  the  disease.  The  mallein  test  and  later  the  agglutination 
test  have  been  and  are  at  present  in  general  use,  but  neither  of  these 
is  sufficiently  reliable  to  be  entirely  satisfactory.  Schiitz  and  Schu- 
bert, German  investigators,  recently  called  attention  to  the  value  of 
complement  fixation  as  affording  a  more  reliable  method  of  diag- 
nosing glanders,  and  within  the  past  year  this  method  has  been 
carefully  studied  and  tested  in  the  Pathological  Division  of  this 
bureau. 

It  will  be  seen  from  the  details  given  in  the  accompanying  paper 
that  the  authors  have  found  the  complement  fixation  test  to  be  highly 
reliable  as  a  diagnostic  agent  for  glanders,  and  they  present  a  thor- 
ough exposition  of  the  method  resulting  from  their  searching  experi- 
ments, including  practical  tests  in  a  recent  outbreak  of  glanders  at 
Washington,  D.  C. 

In  view  of  the  great  economic  and  scientific  importance  of  the 
subject,  and  as  no  work  upon  this  new  method  has  so  far  been  pub- 
lished in  the  United  States,  I  recommend  the  immediate  publication 
of  the  paper  in  the  bulletin  series  of  this  bureau,  in  order  that  the 
value  of  the  method  and  the  technique  necessary  for  its  application 
may  be  made  more  fully  available  in  this  country. 
Respectfully, 

A.  D.  Melvin, 

Chief  of  Bureau. 
Hon.  James  Wilson, 

Secretary  of  Agriculture, 

3 


CONTENTS 


Page. 

Introduction 5 

Hemolysis 7 

Method  of  obtaining  hemolytic  amboceptor  (rabbit  serum ) 9 

Titration  of  hemolytic  rabbit  serum 12 

Method  of  obtaining  complement  (guinea-pig  serum) 14 

Titration  of  complement 14 

Specific  complement  fixation  (deviation) 16 

Method  of  obtaining  serum  of  animals  to  be  tested 18 

Inactivating  the  serum 19 

Preparation  of  the  antigen  (glanders  bacilli  extract) 20 

Titration  of  the  extract 21 

The  complement-fixation  test 23 

Application  of  the  test 24 

Controls 25 

Interpreting  results  of  tests 27 

Controlling  glanders  in  an  infected  stable 29 

Results  of  practical  tests  with  complement  fixation 29 


ILLUSTRATIONS. 


Page. 

Plate  I.  Diagrammatic  representation  of  complement  fixation 8 

II.  Titration  of  hemolytic  amboceptor  (rabbit  serum) 12 

III.  Titration  of  complement  (guinea-pig  serum) 16 

IV.  Titration  of  antigen  (glanders  bacilli  extract) 20 

V.  Final  test  showing  positive  reaction  to  glanders 26 

4 


THE  DIAGNOSIS  OF  GLANDERS  BY  COMPLEMENT 

FIXATION. 


INTRODTJCTIOIT. 

The  early  diagnosis  of  glanders  constitutes  one  of  the  most  im- 
portant and  difficult  tasks  which  confronts  the  veterinarian  engaged 
in  sanitary  work.  This  of  course  does  not  apply  to  the  clinical  cases 
of  glanders,  as  in  such  cases  the  diagnosis  is  usually  made  without 
much  difficulty  from  the  characteristic  symptoms  and  lesions  present. 
In  those  instances,  however,  where  there  are  no  positive  indications 
of  the  disease,  it  is  impossible  to  establish  a  diagnosis  by  physical 
examination,  and  only  through  the  aid  of  some  special  diagnostic 
method  or  test  can  there  be  any  hope  of  determining  the  presence  or 
absence  of  the  disease.  Horses  ajffected  Avith  occult  or  latent  glanders, 
and  in  which  the  disease  is  not  suspected,  are  undoubtedly  great  fac- 
tors in  the  propagation  of  the  infection.  Indeed,  there  are  many 
glandered  horses  which  do  not  show  positive  symptoms  until  the 
later  stages  of  the  disease. 

Since  the  discovery  of  the  glanders  bacillus  in  1883  by  Loeffler  and 
Schiitz  the  diagnosis  of  glanders  has  been  the  subject  of  numerous 
investigations,  and  as  a  result  great  progress  has  been  made  in  its 
determination.  After  the  isolation  of  the  infective  agent  of  the 
disease  the  diagnosis  was  confined  to  the  demonstration  and  culti- 
vation of  the  organism,  or  to  the  reproduction  of  the  disease  by  inoc- 
ulations of  exudates  or  parts  of  diseased  organs  from  affected  horses 
into  susceptible  animals. 

The  first  important  step  toward  determining  obscure  and  latent 
cases  of  glanders  was  made  by  the  discovery  of  mallein.  With  the 
aid  of  this  biological  product  of  the  Bacillus  mallei  a  large  propor- 
tion of  latent  and  occult  cases  of  glanders  can  be  diagnosed,  partic- 
ularly when  such  tests  are  made  by  efficient  and  experienced  veteri- 
narians. There  are,  however,  a  considerable  number  of  glanderous 
animals  in  which  the  mallein  fails  to  give  a  typical  reaction,  and,  on 
the  contrary,  a  reaction  may  follow  the  injection  of  mallein  in  the 
absence  of  glanders.  Thus  mallein  is  not  an  entirely  reliable  diag- 
nostic agent  for  determining  glanders,  nor  has  it  ever  been  considered 

5 


6  DIAGNOSIS   OF   GLANDERS. 

as  efficacious  in  the  detection  of  this  disease  as  tuberculin  for  the 
diagnosis  of  tuberculosis. 

With  the  application  of  the  agglutination  test  for  glanders  it  ap- 
peared that  a  more  satisfactory  method  had  been  found  for  the  diag- 
nosis of  all  types  of  infection  with  this  disease.  It  was  first  sug- 
gested by  McFadyean  in  1896,  after  this  investigator  had  observed 
the  value  of  Widal's  typhoid-fever  agglutination  test,  but  was  not 
generally  adopted  until  the  method  was  perfected  by  Schiitz  and 
Meissner,  whose  interesting  results  were  published  in  1905.  This  test 
has  since  been  extensively  employed  in  practically  every  country 
where  glanders  exists,  and  therefore  ample  opportunity  has  been 
furnished  for  drawing  conclusions  relative  to  its  diagnostic  value. 

While  there  is  no  doubt  that  the  agglutination  test  is  of  great  value 
in  all  cases  of  recent  infection,  the  blood  in  such  cases  possessing  a 
very  high  agglutination  power  (1  to  1,000  and  higher),  nevertheless 
extensive  experience  has  proved  that  horses  affected  with  chronic 
glanders  give  occasionally  a  very  low  agglutination  value,  which  in 
some  cases  is  even  lower  than  that  of  normal  blood  serum  (1  to  400 
or  even  lower).  From  this  condition  it  appears  evident  that  in  cer- 
tain cases  of  chronic  glanders  the  disease  can  be  determined  only  by 
repeated  tests,  and  a  diagnosis  in  such  cases  is  only  possible  from  the 
fluctuation  of  the  agglutination  value — either  an  increase  or  a  de- 
crease— as  it  is  a  well-known  fact  that  this  value  remains  stationary 
in  normal  horses. 

Besides  this  difficulty,  there  should  also  be  taken  into  consideration 
the  fact  that  the  blood  of  normal  horses  sometimes  shows  a  high 
agglutination  value  (1  to  800  and  higher),  and  that  changes  in  the 
agglutination  power  have  been  observed  even  in  animals  free  of 
glanders.  Furthermore,  repeated  agglutination  tests  require  con- 
siderable time,  as  at  least  two  weeks  should  elapse  between  two  tests. 
Therefore  the  agglutination  test  alone  does  not  constitute  an  entirely 
satisfactory  diagnostic  method  for  glanders.  However,  as  its  great 
value  has  been  proved  beyond  doubt  in  the  early  cases  of  infection, 
it  may  well  be  utilized  as  an  adjunct  to  any  other  test  which  may 
be  applied  in  connection  with  the  diagnosis  of  suspected  cases  of  the 
disease. 

Hutyra  compared  the  agglutination  test  with  the  mallein  test  from 
the  tables  included  in  the  works  of  Schiitz  and  Miessner  and  of 
Nevermann,  and  came  to  the  conclusion  that  the  application  of  the 
agglutination  test  alone  has  not  decreased  the  number  of  faulty 
diagnoses.  He  believes  that  the  principal  difference  in  the  results  lies 
in  the  fact  that  a  large  number  of  horses  which  were  classified  as 
only  suspicious  by  the  mallein  test  are  considered  as  actually  in- 
fected by  the  agglutination  test. 


HEMOLYSIS.  7 

In  further  efforts  to  find  a  method  by  which  an  early  diagnosis 
of  glanders  could  be  made,  various  investigators  directed  their  atten- 
tion to  the  precipitation  reaction.  This  is  based  upon  the  fact  that 
when  blood  serum  comes  in  contact  with  a  concentrated  extract  of 
glanders  bacilli  the  precipitins  or  receptors,  which  are  formed  in  the 
blood  of  infected  animals  from  the  time  the  infection  first  occurs,  are 
bound  to  the  bodies  in  the  bacillary  extract,  producing  a  precipita- 
tion which  is  manifested  by  cloudiness  at  the  point  of  contact  of  the 
two  fluids.  This  method  of  diagnosing  glanders  has  recently  been 
recommended  by  Pfeiler  ^  in  Germany  and  by  Konew  ^  in  Eussia, 
but  it  has  not  been  applied  extensively  in  practice.  This  is  probably 
due  to  the  fact  that  the  reading  of  the  reaction  is  in  some  cases  diffi- 
cult, due  to  the  indistinct  ring  which  occasionally  is  formed  at  the 
line  of  contact  between  the  precipitant  and  the  serum. 

In  1909  Schiitz  and  Schubert  ^  published  the  results  of  their  im- 
portant work  on  the  application  of  the  method  of  complement  fixa- 
tion for  the  diagnosis  of  glanders.  And  since  their  experiments 
were  followed  by  splendid  results,  exceeding  by  far  the  results  ob- 
tained from  either  the  mallein  or  the  agglutination  test,  they  recom- 
mended that  this  method  of  diagnosis  in  combination  with  the  agglu- 
tination test  be  taken  as  the  official  test  in  Germany.  This  method, 
overcoming  as  it  does  the  disadvantages  of  the  mallein  and  aggluti- 
nation tests,  constitutes  without  doubt  the  most  reliable  method  for 
the  diagnosis  of  glanders  which  we  have  at  our  command  at  the 
present  time.  The  complement-fixation  test  is,  in  fact,  the  most 
definite  method  known  for  determining  specific  infections  and  is  as 
nearly  perfect  as  a  biological  test  can  be.  It  has  only  recently  been 
introduced  in  veterinary  science  and  the  publications  concerning  it 
are  at  present  limited  exclusively  to  foreign  periodicals.  The  prin- 
ciple of  this  test  is  presented  in  the  phenomenon  of  hemolysis,  which 
was  first  discovered  and  studied  by  Bordet  and  Gengou,  and  extended 
by  Ehrlich,  Morgenroth,  and  Sachs. 

HEMOLYSIS. 

It  is  a  well-known  fact  that  if  red  blood  corpuscles  of  one  animal 
are  introduced  into  another  of  a  different  species  the  blood  of  the 
latter  acquires  the  power  to  dissolve  the  blood  corpuscles  of  the 

1  Pfeiler,  Willy.  Die  Ermlttelung  der  Rotzkrankhelt  durch  die  Prazipltationsmethode. 
Archlv  filr  Wlssenschaftllche  und  Praktlsche  Tlerhellkunde.  Band  35,  Heft  4/5,  pp. 
323-337.     June  24,  1909. 

2  Konew,  D.  Prazipitatlonsreaktlon  als  diagnostlsche  Methode  beim  Rotz.  Voriauflge 
Mlttellung.  Centralblatt  flir  Bakteriologie.  Abt.  1,  Orlg.,  Band  55,  Heft  3,  pp.  251-253. 
July  9,  1910. 

3  Schiitz  and  Schubert.  Die  Ermlttelung  der  Rotzkrankhelt  mit  Hilfe  der  Komplement- 
ablenkungsmethode.  Archlv  fiir  Wlssenschaftllche  und  Praktlsche  Tlerhellkunde.  Band 
35,  Heft  1/2,  pp.  44-83.     1909. 


8  DIAGNOSIS   OP  GLANDERS. 

former  when  mixed  with  them  in  a  reagent  glass.  This  reaction  is 
termed  hemolysis,  which  means  the  dissolution  of  blood  corpuscles, 
thereby  setting  the  hemoglobin  free  in  the  medium  in  which  the 
corpuscles  are  suspended. 

To  illustrate  this  phenomenon,  if  a  rabbit  is  injected  intraperi- 
toneally,  intravenously,  or  subcutaneously  with  washed  red  blood 
corpuscles  of  a  sheep,  the  blood  of  the  rabbit  will  develop  antibodies 
which  possess  a  dissolving  action  for  the  sheep  blood  corpuscles; 
that  is,  the  rabbit  blood  will  contain  specific  hemolysins. 

The  acquired  hemolytic  property  of  the  blood  depends  on  two 
substances.  One  of  these  is  present  in  the  blood  of  every  animal, 
and  is  known  as  the  complement.  It  is  thermo-labile,  which  means 
that  it  is  rendered  inactive  after  the  blood  or  serum  has  been  heated 
to  56°  C.  for  half  an  hour.  The  other  body,  which  is  formed  as  a 
result  of  the  injection  of  blood  corpuscles,  is  thermo-stabile ;  that  is, 
it  resists  heating  even  higher  than  56°  C..  and  is  known  as  immune 
body,  fixative,  sensitizer,  or  hemolytic  amboceptor.  The  name  am- 
boceptor is  derived  from  the  fact  that  it  has  an  affinity  on  the  one 
hand  for  the  blood  corpuscles  of  the  species  of  animal  with  which 
the  animal  has  been  injected,  and  on  the  other  for  the  complement, 
this  union  taking  place  only  after  the  first-mentioned  affinity  has 
been  satisfied. 

These  two  substances,  together  with  the  corpuscles  to  be  dissolved, 
comprise  the  hemolytic  system,  and  their  combination  leads  to 
hemolj'sis.  (See  PI.  I,  A.)  This  means  that  an  opaque  suspension 
of  blood  corpuscles  is  rendered  semitransparent  or  "laked."  The 
hemolysis,  strictly  speaking,  does  not  represent  a  complete  solution, 
but  only  an  action  of  the  hemolj^sin  on  the  stroma  of  the  erythrocytes, 
which  permits  the  escape  of  the  hemoglobin  of  the  red  blood 
corpuscles. 

The  injection  of  blood  corpuscles  of  one  animal  into  another  of  a 
different  species  gives  rise  to  the  development  of  antibodies  which 
confer  upon  the  blood  serum  the  hemolytic  action.  This  phenomenon 
is  somewhat  similar  to  the  production  of  receptors  in  the  formation 
of  antitoxins  which  are  thrown  off,  but  these  receptors  alone  are  not 
able  to  dissolve  the  red  blood  corpuscles,  requiring  also  the  presence 
of  a  ferment.  This  ferment,  however,  is  a  constant  constituent  of  the 
blood  and  is  known  as  the  complement. 

That  both  of  these  substances  are  constantly  present  in  the  hemo- 
lytic serum  can  be  demonstrated  in  the  following  manner:  If  the 
hemolytic  serum  is  heated  to  56°  C.  for  half  an  hour,  thereby  de- 
stroying the  complement,  this  serum  will  no  longer  possess  a  hemo- 
lytic action;  that  is,  it  will  no  longer  dissolve  red  blood  corpuscles. 
This  heating  of  the  serum  is  known  as  inactivation.  On  the  other 
hand,  if  to  such  inactivated  serum  there  be  added  fresh  untreated 


BuL.  136,  Bureau  of  Animal  Industry,  U.  S.  Dept.  of  Agriculture. 


Plate  I. 


A.  (■  A  +  r=2  = 


rjSheea) 


^p)  one/  o/nAoc€a^r 

f  (no  nemo/usis). 


B. 


Sacfer/o 


M^Mrf'o/uti'c    union of^Sk6atr^r/a  ^^^ 

>6acef>^r.  on^/^^amiocc/aiiir:  Comp/cmGnt: 

(/Vo  6octer/'o/(js/.3) 


<?f^€  scrum  ^ftAi 
•^ty/ont/ers 

(GhntJei^ 
/a/r//yefi). 


n  ^-f 

^  4i>!f/,f/anc/ers      tomplemenr 

omoocep/di:s- 
Jjottena.  ' 

(G/anc/ens 


Diagrammatic  Representation  of  Complement  Fixation. 


A,  Hemolytic  system. 

B,  Bacteriolytic  system. 

C,  Negative  reaction  with  normal  horse  serum. 

D,  Positive  reaction  with  glandered  horse  serum. 


METHOD  OF  OBTAINING  THE  HEMOLYTIC  AMBOCEPTOR.  9 

serum,  which  in  itself  has  no  hemolytic  properties,  hemolysis  will 
result.  Thus  by  the  addition  of  this  fresh  serum  a  reactivation  is 
accomplished.  This  is  explained  by  the  fact  that  through  the  heat- 
ing of  the  serum  one  of  the  substances  necessary  for  the  hemolysis 
has  been  destroj^ed,  which  is  the  complement.  After  the  complement 
has  been  destroyed  by  heating  it  can  be  replaced  by  the  addition  of 
any  normal  serum,  because  it  is  known  that  the  complement  is  present 
in  all  blood.  However,  the  guinea-pig  serum  appears  to  be  the  most 
satisfactory  in  the  application  of  hemolysis,  inasmuch  as  it  is  very 
rich  in  complement  and  only  a  very  small  quantity  is  required  to  be 
added  to  the  inactivated  hemolytic  serum  in  order  to  produce 
hemolysis. 

Accordingly,  the  substances  necessary  for  hemolysis  are  (1)  the 
hemolytic  amboceptor,  which  is  the  serum  of  a  rabbit  that  has  been 
injected  with  washed  sheep  blood  corpuscles,  (2)  the  complement 
in  the  form  of  normal  guinea-pig  serum,  and  (3)  washed  red  blood 
corpuscles  of  a  sheep.  In  the  preparation  of  these  different  substances 
it  is  necessary  to  fix  standards  of  practical  constancy  by  proceed- 
ing along  definite  lines  in  the  following  manner : 

METHOD   OF   OBTAINING   THE    HEMOLYTIC    AMBOCEPTOR    (rABBIT   SERUm). 

Strong,  vigorous  rabbits  are  selected,  and  they  are  injected  in- 
traperitoneally  with  a  suspension  of  washed  red  blood  corpuscles 
from  a  sheep.  Three  injections  are  made  at  intervals  of  seven  days 
with  7  c.  c,  10  c.  c,  and  12  c.  c.  of  these  blood  corpuscles  suspended 
in  like  quantities  of  phj-^siological  salt  solution.^ 

The  sheep  blood  is  obtained  by  bleeding  a  vigorous  sheep  from 
the  jugular.  The  side  of  the  neck  is  clipped  and  shaved,  and  the 
part  over  the  jugular  disinfected  with  75  per  cent  alcohol.  Then  a 
sterilized  small-calibered  trocar  is  inserted  into  the  jugular,  and  the 
blood  is  collected  in  a  sterile  bottle  containing  a  few  glass  beads. 
After  the  desired  quantity  of  blood  is  obtained  it  is  shaken  for  10 
minutes  in  order  to  defibrinate  it.  After  defibrination,  it  is  filtered 
through  a  double  layer  of  sterile  gauze  into  the  glass  tube  in  which 
the  washing  is  to  take  place.  The  glass  tube  containing  the  blood 
is  then  filled  with  salt  solution  and  placed  in  a  centrifuge  which  has 
a  speed  of  2,500  to  3,000  revolutions  per  minute.  After  the  red  blood 
corpuscles  are  thrown  down  the  clear  fluid  above  the  corpuscles  is 
pipetted  off.  Then  the  blood  corpuscles  are  again  thoroughly  mixed 
with  salt  solution  in  the  proportion  of  1  to  9,  and  the  centrifugaliza- 
tion  is  repeated.  This  washing  should  be  carried  out  three  or  even 
four  times  in  order  to  eliminate  all  the  serum  adhering  to  the  red 

1  Unless  otherwise  stated,  the  term  "  salt  solution  "  In  this  bulletin  I'efers  to  ao  0.85  per 
cent  solution  of  sodium  chlorid. 

10040°— Bull.  136—11 2 


10  DIAGNOSIS   OF   GLANDERS. 

blood  corpuscles.  Such  washed  blood  corpuscles  can  then  be  used  for 
the  injection  of  rabbits  in  the  preparation  of  hemolytic  amboceptors, 
as  well  as  for  the  test  proper. 

The  washing  of  the  sheep  blood  corpuscles  must  be  thoroughly 
carried  out,  inasmuch  as  the  presence  of  even  traces  of  serum  adher- 
ing to  the  corpuscles  may  cause  difficulty  in  obtaining  satisfactory 
results.  If  rabbits  were  injected  with  red  blood  corpuscles  contain- 
ing a  small  quantity  of  serum,  the  rabbits  would  develop,  not  only 
antibodies,  or  immune  bodies,  but  also  coagulins  and  anticomple- 
ments,  and  the  presence  of  these  substances  would  give  rise  to  diffi- 
culties in  demonstrating  the  presence  or  absence  of  a  complete  hemol- 
ysis. Furthermore,  if  blood  corpuscles  containing  even  traces  of 
serum  were  used  in  the  tests  it  might  produce  a  fixation  of  the  com- 
plement and  thereby  give  rise  to  errors.  Such  errors  would  occur 
particularly  if  the  hemolytic  action  of  the  rabbit  serum  was  not 
very  high. 

The  washed  blcfod  corpuscles  should  be  used  for  the  injection  of 
the  rabbit  on  the  day  the  blood  is  drawn.  For  testing  purposes, 
however,  it  will  keep  for  two  or  three  days  in  the  ice  chest. 

The  rabbits  to  be  injected  are  shaved  on  the  posterior  part  of  the 
abdomen,  and  the  skin  is  disinfected  with  75  per  cent  alcohol.  They 
are  then  held  with  the  head  doAvn  by  an  assistant  in  order  to  prevent 
the  puncturing  of  the  intestines.  The  blood  corpuscles  to  be  in- 
jected are  mixed  with  an  equal  quantity  of  salt  solution  and  heated 
in  a  water  bath  to  body  temperature.  Intravenous  injections  have 
been  recommended  by  some  investigators,  but  it  was  found  in  our 
work  that  intraperitoneal  injections  gave  very  satisfactory  results, 
and  furthermore,  there  is  very  little  danger  of  losing  rabbits  from 
various  complications  by  this  method. 

After  three  injections  with  the  quantities  and  at  the  intervals 
stated  above,  a  small  amount  of  blood  is  taken  from  the  rabbit  on  the 
fifth  or  sixth  day  after  the  last  injection.  This  blood  is  then  titrated 
in  order  to  determine  whether  its  hemolytic  action  is  of  sufficient 
strength  for  future  work.  If  the  blood  serum  is  found  to  be  of  a 
sufficiently  high  titre,  the  rabbit  may  be  either  bled  to  death,  or. 
what  is  far  more  satisfactory,  about  15  c.  c.  of  blood  may  be  drawn 
from  the  veins  of  the  ear.  By  the  latter  method  the  rabbit  may  be 
used  continuously  for  the  production  of  hemolytic  serum.  If  it  is 
desired  to  obtain  the  blood  by  bleeding  the  rabbit  to  death,  the 
animal  is  anesthetized  by  a  mixture  of  chloroform  and  ether,  the  hair 
f)n  the  neck  is  shaved,  the  skin  is  disinfected,  and  an  incision  made 
'on  one  side  of  the  neck  in  order  to  sever  both  the  jugular  vein  and 
the  carotid  artery.  Should  the  flow  of  blood  cease  on  this  side,  the 
other  side  may  also  be  cut.     The  blood  is  collected  in  centrifuge 


METHOD  OF  OBTAINING  THE  HEMOLYTIC  AMBOCEPTOR.  11 

tubes,  and  after  the  bleeding  has  been  completed  centrifugalization 
of  the  blood  is  accomplished  and  the  supernatant  serum  drawn  off 
with  a  pipette. 

Bleeding  of  the  rabbit  through  the  veins  of  the  ear  is  best  accom- 
plished in  the  following  way:  After  washing  the  ear  and  closely 
clipping  the  hair  over  the  veins  on  the  outside  of  the  ear,  the  skin 
is  disinfected  with  75  per  cent  alcohol.  Then  a  pledget  of  cotton  is 
soaked  in  hot  water  (about  45°  or  50°  C.)  and  wound  around  the 
base  of  the  ear  in  order  to  produce  a  hyperemia  of  the  blood  vessels. 
When  a  sufficient  dilation  of  the  vessels  is  observed',  the  middle  and 
posterior  auricular  veins  are  severed,  and  the  blood  is  then  collected 
in  centrifuge  tubes.  If  the  blood  ceases  to  flow,  the  cotton  should  be 
removed  from  the  base  of  the  ear,  and  after  placing  it  in  hot  water 
it  is  again  applied  to  the  ear  and  the  hyperemia  is  thereby  reestab- 
lished. In  case  the  coagulation  of  the  blood  has  prevented  its  flow 
at  the  place  where  the  veins  were  severed,  the  wound  may  be  scraped 
with  a  knife,  and  usually  the  blood  will  commence  to  flow  freely 
again.  The  collected  blood  is  then  treated  in  the  same  manner  as 
described  above — that  is,  centrifuged — and  the  supernatant  serum 
pipetted  off. 

Should  this  hemolytic  rabbit  serum  be  used  before  it  is  3  days  old, 
it  must  be  inactivated  by  heating  to  56°  C.  for  one-half  hour.  After 
this  time  the  hemolytic  rabbit  serum  is  preserved  with  one-half  of 
1  per  cent  of  carbolic  acid — that  is,  to  each  9  c.  c.  of  serum  1  c.  c. 
of  a  5  per  cent  carbolic-acid  solution  is  added.  Serum  preserved  in 
this  way  may  be  kept  for  two  or  three  months.  However,  it  should  be 
retitrated  every  two  or  three  weeks,  as  occasionally  the  titre  of  the 
serum  drops.  Such  carbolized  hemolytic  serum  does  not  require 
inactivation. 

\Vhen  preparing  hemolytic  serum  it  is  best  to  start  with  several 
rabbits,  as  occasionally  one  may  die  as  a  result  of  anaphylaxis,  and, 
again,  some  rabbits  are  not  adapted  for  the  production  of  hemolysins. 

The  titre  of  the  hemolj'tic  serum  from  a  rabl)it  does  not  remain 
stationary,  but  for  two  or  three  weeks  after  the  last  injection  it 
gradually  lowers  until  it  reaches  that  of  a  normal  animal.  If,  how- 
ever, such  a  rabbit  is  reinjected  with  washed  corpuscles  after  several 
weeks,  the  hemolysins  will  again  appear  after  a  short  time.  Hemo- 
lysins are  kept  more  or  less  in  reserve  in  the  cells  of  such  an  animal. 
The  renewed  injection  acts  as  a  stimulant,  and  these  bodies  are 
quickly  thrown  off  into  the  blood,  while  in  a  normal  animal  they 
form  slowly,  fiist  being  formed  in  the  cells.  Thus,  the  advisability 
of  keeping  rabbits  which  have  been  bled  from  the  veins  of  the  ear 
for  further  production  of  hemolytic  amboceptors  can  readily  be  seen. 


12 


DIAGNOSIS   OF  GLANDERS. 


TITRATION   OF  HEMOLYTIC   BABBIT   SEBUM. 

The  hemolytic  rabbit  serum  is  titrated  in  order  to  establish  the 
smallest  quantity  of  serum  that  will  produce  hemolysis  in  the  pres- 
ence of  a  certain  quantity  of  complement  and  the  suspension  of 
washed  blood  corpuscles  of  a  sheep.  The  amount  of  hemolytic  serum 
that  will  produce  complete  hemolysis  in  two  hours  at  37°  C.  is  an 
amboceptor  unit. 

.For  the  preliminary  work  of  titration  10  test  tubes  are  taken,  and 
dilutions  of  the  hemolytic  serum  are  made  with  salt  solution  in 
proportions  of  1  to  100,  200,  400,  500,  etc.,  up  to  4,000.  These  dilu- 
tions are  made  from  basic  dilutions  of  1  to  100  and  1  to  1,000  as 
may  be  seen  from  Table  1. 

Table  1. — Titration  of  rabbit  serum  (hemolytic  amboceptor). 


Tube. 

NaCl 
solution.' 

Amboceptor. 

Comple- 
ment.2 

Blood  cor- 
puscles.' 

Remarks. 

1 
2 
3 
4 
5 
6 
7 
8 
9 
10 

c.  c. 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 
2.5 

1  c.  c.  of— 

1:   100 

1:   200 

1:   400 

1:  500 

1:   600 

1:   800 

1:1,000 

1:1,500 

1:2,000 

1:4,000 

c.  c. 
0.5 
.5 
.5 
.5 
.5 
.5 
.5 
.5 
.5 
.5 

c.  c. 

11 

3.5 
3.0 
4.0 

.5 

. 

Complement  control  (no  hemolysisshould  occur). 

12 

1:   100 

Amboceptor  control  (no  hemolysis  should  occur). 

13 

Salt-solution  control  (no  hemolysisshould  occur). 

1  0.85  per  cent  NaCl  solution. 

^0.5  c.  c.  of  a  10  per  cent  solution  of  the  complement. 

^5  per  cent  suspension  of  sheep-blood  corpuscles  washed  in  salt  solution. 

From  each  batch  of  serum  taken  from  the  rabbit  a  basic  dilution  is 
made  in  the  proportion  of  1  to  100,  200,  400,  etc.,  up  to  4,000.  From 
these  basic  dilutions  the  third  column  in  Table  1  is  made,  as  indicated 
below : 


A  dilution  of — 

200  is  made  by  adding  1  c.  c. 

400  is  made  by  adding  1  c.  c. 

500  is  made  by  adding  1  c.  c. 

600  is  made  by  adding  1  c.  c. 

800  is  made  by  adding  1  c.  c. 
1,000  is  made  by  adding  1  c.  c. 
1,500  is  made  by  adding  2  c.  c. 
2,000  is  made  by  adding  1  c.  c. 
4,000  is  made  by  adding  1  c.  c. 

It  will  be  observed  that  the 
dilution. 


of  1  to  100  dilution  to  1  c.  c.  NaCl  solution, 
of  1  to  100  dilution  to  3  c.  c.  NaCl  solution, 
of  1  to  100  dilution  to  4  c.  c.  NaCl  solution. 
of  1  to  100  dilution  to  5  c.  c.  NaCl  solution, 
of  1  to  100  dilution  to  7  c.  c.  NaCl  solution, 
of  1  to  100  dilution  to  9  c.  c.  NaCl  solution, 
of  1  to  1,000  dilution  to  1  c.  c.  NaCl  solution, 
of  1  to  1,000  dilution  to  1  c.  c.  NaCl  solution, 
of  1  to  1,000  dilution  to  3  c.  c.  NaCl  solution. 

last  three  are  made  from  the  1  to  1,000 


BuL.  136,  Bureau  of  Animal  Industry,  U.  S.  Dept.  of  Aqriculture. 


Plate  II 


o 


o 


TITRATION    OF    HEMOLYTIC    RABBIT    SERUM.  13 

The  titration  proper  is  then  made  in  the  following  manner :  Ten 
additional  test  tubes  are  each  filled  with  2.5  c.  c.  of  salt  solution,  to 
which  is  then  added  the  hemolytic  serum  (amboceptor)  in  quantities 
of  1  c.  c.  of  the  different  dilutions  to  each  tube.  Thus,  in  the  first 
tube  we  add  1  c.  c.  of  the  dilution  of  1  to  100 ;  in  the  next,  1  c.  c.  of 
the  dilution  of  1  to  200;  and  in  the  third,  1  c.  c.  of  the  dilution  of  1  to 
400,  etc.  Afterwards  the  complement  of  the  guinea-pig  serum  is 
added  in  quantities  of  0.5  c.  c.  of  a  10  per  cent  solution  to  each  tube, 
and  finally  1  c.  c.  of  a  5  per  cent  suspension  of  sheep  corpuscles  in  salt 
solution  is  placed  in  each  tube. 

Besides  these  10  tubes,  there  are  also  three  control  tubes,  one  to 
show  that  the  complement  alone  (without  the  amboceptor)  will  not 
produce  hemolysis,  tlie  second  that  the  amboceptor  alone  without  the 
complement  will  not  produce  hemolysis,  and  the  third  that  the  salt 
solution  alone  will  not  produce  hemolysis.  Thus,  in  the  first  control 
tube  we  add  3.5  c.  c.  of  salt  solution,  0.5  c.  c.  complement,  and  1  c.  c. 
suspension  of  sheep  corpuscles.  In  the  second  control  tube  we  add 
3  c.  c.  salt  solution,  1  c.  c.  amboceptor  of  the  1  to  100  dilution,  and 
1  c.  c.  suspension  of  sheep  corpuscles.  In  the  third  control  tube  we 
add  4  c.  c.  salt  solution  and  1  c.  c.  sheep  corpuscles.  It  is  advisable  to 
place  the  tubes  for  the  titration  test  in  the  lower  row  and  the  control 
tubes  in  the  upper  row  of  the  test-tube  rack. 

As  can  be  seen  from  the  various  quantities  added  to  the  tubes,  the 
final  volume  is  always  uniformly  5  c.  c.  Thus,  the  different  amounts 
of  the  blood  derivatives  used  are  always  made  up  to  5  c.  c.  by  the 
addition  of  salt  solution. 

After  adding  the  substances  in  the  test  tubes  in  the  order  given,  the 
tubes  are  w^ell  shaken,  placed  in  a  rack,  and  the  rack  put  in  an  incu- 
bator at  37°  C.  for  two  hours.  Then  it  is  removed  from  the  incubator 
and  the  results  are  read.     (See  PI.  11.) 

The  highest  dilution  in  which  complete  hemolysis  has  taken  place 
represents  the  titre  of  the  hemolytic  amboceptor.  Thus,  if  complete 
hemolysis  has  taken  place  in  the  tubes  where  the  dilution  used  in  the 
rabbit  serum  was  1  to  2,000,  the  hemolytic  amboceptor  of  that  serum 
is  represented  by  2,000.  This  titre,  however,  is  not  used  in  the 
glanders  test,  but  rather  its  double  strength,  which  would  be  1  to  1,000. 

The  titre  of  the  hemolytic  amboceptor  for  use  in  glanders  diagnosis 
should  not  be  less  than  1  to  1,000,  and  therefore  if  the  rabbit  serum 
should  prove  of  a  low^er  titre  it  should  not  be  used  for  this  test.  A 
low  titre  of  the  hemolytic  amboceptor  would  disturb  the  results  of 
the  test,  inasmuch  as  in  such  low  dilutions  too  much  serum  would 
have  to  be  used,  and  as  the  serum  contains  other  substances  in  addi- 
tion to  amboceptors  it  would  have  an  influencing  effect  on  the 
hemolysis. 


14  DIAGNOSIS   OF  GLANDERS. 

It  is  advisable  to  preserve  the  carbolized  hemolytic  amboceptor  in 
small  vials  containing  1  to  2  c.  c.  of  the  rabbit  serum,  sealing  them 
with  paraffin.  By  this  procedure  the  contents  of  a  vial  are  used  up 
more  quickly  and  without  frequent  exposure. 

The  titration  test  of  the  hemolytic  amboceptor  is  given  in  consecu- 
tive order  in  Table  1,  and  the  method  of  carrying  out  the  tests  relative 
to  the  addition  of  the  different  substances  is  likewise  given  in  con- 
secutive order  in  all  the  tables.  Thus  the  number  of  test  tubes  used  is 
indicated,  as  well  as  the  various  substances  and  the  quantities  to  be 
added,  as  they  follow  each  other.  It  is  our  opinion  that  by  this 
method  the  description  of  the  tests  can  be  followed  without  much 
difficulty,  and  by  keeping  these  tables  in  the  foreground  the  tests 
themselves  may  be  applied  readily,  even  by  those  who  have  had  but 
little  experience  in  this  work.  Of  course  accuracy  in  technique  is  the 
most  important  factor  in  the  success  of  this  line  of  serum  diagnosis, 
and  too  much  emphasis  can  not  be  laid  upon  this  fact. 

METHOD   or   OBTAINING   COMPLEMENT    ( GUINEA-PIG    SERUM ). 

The  complement  represents  the  blood  serum  of  a  healthy  guinea  pig, 
and  is  obtained  by  bleeding  the  animal  by  severing  the  carotid  and 
the  jugular. 

The  guinea  pig  is  anesthetized  with  a  mixture  of  chloroform  and 
ether,  and  the  neck  is  shaved  and  then  disinfected  with  a  75  per  cent 
solution  of  alcohol.  The  animal  is  held  by  an  assistant  with  its  head 
down,  while  the  operator  uses  his  left  hand  to  pull  taut  the  skin  over 
the  region  of  the  throat  and  his  right  hand  to  make  an  incision  on 
one  side  of  the  neck  by  which  the  carotid  and  the  jugular  are  sev- 
ered. The  centrifuge  tube  is  immediately  held  under  the  opening,  in 
order  to  collect  all  the  blood.  After  the  flow  ceases,  the  same  tech- 
nique is  practiced  on  the  other  side  of  the  neck.  Care  should  be  taken 
to  avoid  the  cutting  of  the  trachea. 

The  blood  thus  collected,  which  usually  amounts  to  10  or  12  c.  c, 
is  placed  in  the  ice  chest,  and  after  one  or  two  hours  the  serum  sepa- 
rates from  the  clot.  The  serum  is  then  drawn  off,  and  the  rest  is 
placed  in  a  centrifugal  machine,  in  order  to  obtain  all  the  serum 
present  in  the  clot.  The  guinea-pig  serum  should  always  be  used 
fresh,  and  it  is  never  advisable  to  use  it  after  the  second  day,  as  the 
complement  becomes  considerably  reduced  upon  standing. 

TITBATION   OF   COMPLEMENT. 

It  is  desirable  to  titrate  the  complement  of  every  guinea  pig,  as 
such  practice  will  insure  more  accurate  work  and  better  results  in 
the  glanders  test.    By  titration  of  the  complement  we  aim  to  estab- 


METHOD   OP   OBTAINING   COMPLEMENT.  15 

lish  the  complement  unit  which  is  the  smallest  quantity  of  comple- 
ment necessary  to  produce  complete  hemolysis  in  the  presence  of  one 
amboceptor  unit  and  a  suspension  of  blood  corpuscles  of  sheep.  The 
smallest  quantity  is  then  taken  as  its  test  value.  For  the  titration  of 
the  complement  six  test  tubes  are  used,  in  addition  to  the  three  for 
controls.  A  10  per  cent  basic  dilution  is  made  of  the  complement 
in  the  first  test  tube;  that  is,  to  2.7  c.  c.  of  salt  solution  0.3  c.  c.  of 
complement  is  added.  From  this  basic  dilution  the  other  dilutions 
are  made  by  consecutively  reducing  the  quantity  of  complement  in 
the  different  test  tubes.  Thus,  in  the  second  test  tube  0.5  c.  c.  of  the 
10  per  cent  basic  dilution  is  added,  in  the  third  0.4  c.  c,  in  the  fourth 
0.3,  and  so  on.  Of  course  we  add  to  each  of  these  test  tubes  sufficient 
quantities  of  salt  solution  to  make  the  required  3  c.  c.  Thus,  into 
the  second  test  tube  we  add  2.5  c.  c,  in  the  third  2.6  c.  c,  etc.,  of  salt 
solution.  After  the  complement  has  been  added  in  quantities  stated 
above,  it  is  followed  by  the  amboceptor.  One  cubic  centimeter  of 
the  hemolytic  amboceptor  of  which  the  titre  has  previously  been  de- 
termined is  added,  and  finally  1  c.  c.  of  a  5  per  cent  emulsion  of  blood 
corpuscles.  The  purpose  of  the  controls  is  to  establish  in  the  first 
that  the  complement  alone  without  the  amboceptor  does  not  produce 
hemolysis;  in  the  second,  that  the  amboceptor  alone  without  the 
complement  produces  no  hemolysis;  and  in  the  third,  that  the  salt 
solution  alone  does  not  produce  hemolysis.  The  first  control  tube 
contains  3.5  c.  c.  salt  solution,  0.5  c.  c.  of  the  10  per  cent  basic  dilu- 
tion, and  1  c.  c.  of  suspension  of  corpuscles;  in  the  second  control  3 
c.  c.  of  salt  solution,  1  c.  c.  of  the  amboceptor  dilution,  and  1  c.  c.  of 
suspension  of  blood  corpuscles;  in  the  third  control,  4  c.  c.  of  salt 
solution  and  1  c.  c.  of  suspension  of  blood  corpuscles  are  used. 

After  shaking  all  the  tubes,  the  rack  is  placed  in  the  incubator  for 
two  hours,  and  removed  in  order  to  read  the  results.  (See  PI.  III.) 
The  highest  dilution  of  complement  in  the  tube  in  which  the  hemolj^- 
sis  is  complete  indicates  the  titre  of  the  complement.  For  instance, 
if  hemolysis  is  complete  in  the  tube  where  0.3  c.  c.  of  the  10  per  cent 
basic  dilution  of  the  complement  has  been  used,  and  hemolysis  is  in- 
complete in  the  tube  in  which  0.2  c.  c.  of  a  10  per  cent  basic  dilution 
has  been  used,  then  the  titre  of  the  complement  is  0.03  c.  c,  inasmuch 
as  we  have  started  with  a  10  per  cent  basic  dilution.  Thus,  in  the 
tests  in  this  instance  it  would  be  necessary  to  use  a  3  per  cent  com- 
plement solution. 

In  Table  2,  the  titration  of  the  complement  is  given  as  in  Table  1 
for  the  hemolytic  amboceptor,  and  this  can  be  followed  without  much 
difficulty.  Particular  care,  however,  should  be  taken  to  use  exaci; 
quantities  as  designated  in  the  table. 


16 


DIAGNOSIS   OF  GLANDERS. 
Table  2. — Titration  of  complement. 


Tube. 

NaCI  so- 
lution. 1 

Comple- 
ment.* 

Ambo- 
ceptor.3 

Blood 
cor- 
puscles.* 

Remarks. 

1 

c.c. 
2.7 

2.6 
2.6 
2.7 

2.8 
2.9 

c.  c. 
0.3 

6.5 

.4 
.3 
.2 
.1 

c.  c. 

c.c. 

Basle  dilution  of  complement. 

Tubes  2  to  6  are  for  establishing  the  smallest  quan- 
tity of  complement  which  produces  complete 
hemolysis.  This  smallest  quantity  is  then  taken 
as  its  test  value.  For  instance,  if  smallest  quan- 
tity Is  0.03,  then  to  97  c.  c.  NaCl  solution  3  c.  c. 
complement  is  added;  if  it  is  0.02,  only  2  c.  c.  is 
added  to  98  c.  c.  NaCl  solution. 

2 
3 
4 

5 
6 

1 
1 
1 
1 

1 

7 

3.5 
3.0 
4.0 

.5 

1 
1 

1 

Complement  control  (no  hemolysis  should  occur). 
Amboceptor  control  (no  hemolysis  should  occur). 
Salt-solution  control  (no  hemolysis  should  occur). 

8 

i 

9 

^  0.85  per  cent  NaCl  solution. 

2  Guinea  pij;  serum  in  diminishing  quantities. 

^  Of  previously  titrated  hemolytic  serum,  double  dissolving  quantity. 

*  5  per  cent  suspension  of  sheep  blood  corpuscles  washed  in  salt  solution. 

5  This  amount  is  taken  from  the  basic  dilution  In  tube  1. 

The  amboceptor  should  be  inactivated  when  used  fresh;  that  is, 
before  it  is  three  days  old.  Otherwise  it  is  carbolized  with  10  per 
cent  of  5  per  cent  carbolic-acid  solution,  and  kept  in  ice  box — in  this 
case  it  is  used  without  inactivation. 

Place  tube  rack  in  incubator  for  2  hours  and  read  results. 

This  test  should  be  made  as  a  preliminary  to  every  suspected 
glanders  serum  test,  as  it  is  always  necessary  to  determine  the  smallest 
quantity  of  complement  to  be  used  for  the  final  test.  Of  course, 
within.  24  hours  any  number  of  tests  can  be  made  with  the  same  com- 
plement dilutions. 

SPECIFIC    COMPLEMENT    FIXATION    (DEVIATION). 

Complement  fixation  is  a  biological  reaction  in  which  the  phenome- 
non of  hemolysis  is  employed  as  the  fundamental  principle.  It  is 
so  called  on  account  of  the  fact  that  the  complement  has  been  fixed 
by  the  combination  of  antigen  with  antibody  and  thus  prevented 
from  participating  in  the  hemolytic  process  in  which  it  is  essential 
in  order  to  have  hemolysis.  By  this  method  even  small  quantities  of 
amboceptors  (antibodies)  can  be  demonstrated  in  a  serum. 

The  presence  of  an  infectious  principle  in  the  organism  of  an 
animal  or  a  man  has  a  stimulating  effect  on  the  production  of  anti- 
bodies (immune  bodies).  If  a  serum  containing  such  immune  bodies 
is  inactivated  and  brought  into  contact  Avith  the  antigen  in  the 
presence  of  complement,  the  complement  will  become  firmly  fixed 
by  the  combined  immune  body  and  antigen.  (See  PI.  I,  B.)  Thus, 
anchoring  takes  place  between  the  antigen  and  the  antibody  in  which 
the  complement  becomes  fixed.  This  anchoring  is  thoroughly  estab- 
lished when  the  mixture  is  placed  in  an  incubator  for  one  hour. 
The  addition  of  the  hemolytic  amboceptor  and  blood  corpuscles  to 


BuL.  136,  Bureau  of  Animal  Industry,  U.  S.  Dept.  of  Aqriculture. 


Plate    III 


SPECIFIC   COMPLEMENT  FIXATION.  17 

such  an  anchored  antigen  and  immune  body  will  have  no  effect.  (See 
PI  I,  D.)  Thus,  no  hemolysis  will  take  place,  inasmuch  as  the  com- 
plement has  been  fixed  bj'^  the  immune  body  and  the  antigen,  thereby 
leaving  the  hemolytic  system  incomplete.  On  the  other  hand,  if  the 
inactivated  serum  contains  no  immune  bodies,  there  would  be  no 
substance  in  the  serum  to  anchor  the  antigen.  As  a  result,  therefore, 
no  fixation  of  complement  will  occur,  this  being  left  free,  and  on 
addition  of  hemolytic  amboceptor  and  blood  corpuscles,  hemolysis 
will  now  take  place.  (See  PI.  I,  C.)  Neither  the  antigen  nor  the 
antibody  alone  can  fix  the  complement  and  thereby  influence  hemo- 
lysis when  the  hemolytic  amboceptor  and  blood  corpuscles  are  added. 
However,  in  combination  the  fixation  will  invariably  take  place,  and 
on  the  addition  of  the  hemolytic  amboceptor  and  blood  corpuscles 
hemolysis  will  not  be  produced. 

Since  the  discovery  of  this  phenomenon  it  has  been  utilized  exten- 
sively in  serum  diagnosis,  but  probably  its  greatest  value  has  been 
obtained  from  the  Wassermann  reaction  for  the  diagnosis  of  syphilis. 
It  has  also  been  employed  in  other  diseases  with  more  or  less  satis- 
faction, and  its  great  field  in  bacteriological  investigations  has  not 
yet  been  exhausted  for  the  practical  diagnosis  and  determination  of 
immune  bodies  in  serum.  In  veterinary  practice  complement  fixa- 
tion is  now  gradually  becoming  used  for  the  diagnosis  of  glanders. 
This  method  of  diagnosing  glanders  has  given  the  most  favorable 
results  in  Germany,  and  constitutes  at  the  present  time  the  official 
test  for  Prussia  and  other  parts  of  Germany.  It  has  also  been  used 
in  the  diagnosis  of  other  diseases  of  animals,  but  not  with  such  suc- 
cess as  in  glanders.  Particularly  in  tuberculosis  the  results  were  not 
uniform  and  otherwise  not  very  promising. 

The  presence  of  the  specific  immune  bodies  (bacteriolytic  ambocep- 
tors) in  the  serum  of  glandered  horses  brings  about  the  fixation  of 
the  complement  when  the  antigen  in  the  form  of  glanders  bacilli 
extract  is  added  to  the  hemolytic  system.  The  serum  of  glandered 
horses,  therefore,  contains  antibodies  (immune  bodies)  against  glan- 
ders bacilli,  which  are  specific  only  for  the  glanders  bacilli  and  for 
no  other  infection.  The  complement  fixation  accordingly  represents 
a  specific  test,  as  only  in  the  presence  of  the  glanderous  immune 
bodies  and  glanderous  antigen  will  the  reaction  take  place.  If,  in- 
stead of  the  glanderous  immune  bodies,  other  antibodies  of  another 
infectious  disease  be  present  in  the  blood  serum,  they  will  exert  no 
effect  whatsoever  on  the  glanderous  antigen ;  and,  on  the  other  hand, 
if  serum  containing  glanderous  immune  bodies  is  brought  in  contact 
with  an  antigen  of  another  infectious  disease,  it  will  also  have  no 
effect  on  the  reaction.  By  this  fixation  of  the  complement  the  hemo- 
lytic system  is  left  incomplete,  and  as  a  result  no  hemolysis  will  take 
place.    This  fixation  of  the  complement  by  the  antigen  and  immune 


18  DIAGNOSIS   OF  GLANDERS. 

bodies  of  glanders  in  the  horse  serum  constitutes  the  diagnostic  test 
for  this  disease. 

In  the  application  of  the  test  it  is  necessary  to  have  substances  con- 
stituting the  hemolytic  system,  which  are  the  washed  blood  corpuscles 
of  a  sheep,  the  hemolytic  amboceptor  (rabbit  serum) ,  and  complement 
(normal  guinea  pig  serum).  The  quantity  of  the  hemolytic  ambo- 
ceptor to  be  used  has  been  established  by  titration  and  described  in  a 
preceding  part  of  this  publication.  In  the  test  for  glanders  the 
double  strength  of  the  determined  titre  of  the  hemolytic  amboceptor 
is  used.  A  slight  excess  in  the  quantity  of  the  amboceptor  does  not 
alter  the  outcome  of  the  tests. 

On  the  other  hand,  the  establishment  of  the  smallest  amount  of 
complement  which  will  produce  hemolysis  in  the  presence  of  the 
hemolytic  amboceptor  and  blood  corpuscles  is  very  essential,  and 
accordingly  this  should  be  established  by  the  previously  described 
preliminary  test,  before  tests  for  glanders  are  undertaken.  By  omit- 
ting this  it  is  possible  that  an  excessive  amount  of  complement  would 
be  used  which  would  in  some  cases  affect  the  final  results  of  the  test. 
An  oversupply  of  complement  in  the  test  may  not  only  prove  sufficient 
to  be  fixed  by  the  immune  bodies  (antibodies)  and  antigen,  but  there 
might  be  also  enough  complement  left  to  produce  an  incomplete  or  an 
almost  complete  hemolysis.  Thus  it  is  evident  that  it  is  of  great 
importance  to  establish  by  such  a  preliminary  test  (see  Table  2)  the 
exact  quantity  of  complement  to  be  used.  Schiitz  and  Schubert  found 
that  the  success  of  this  method  of  diagnosing  glanders  depended 
greatly  upon  the  proper  quantity  of  complement  used,  and  therefore 
the  establishment  of  this  quantity  should  not  be  omitted. 

The  red  blood  corpuscles  of  the  hemolytic  system  always  constitute 
a  uniform  quantity — that  is,  a  5  per  cent  suspension  of  the  washed 
corpuscles  in  salt  solution.  As  has  been  stated,  this  may  be  kept  for 
testing  purposes  for  two  or  even  three  days  in  the  ice  chest.  The 
method  of  obtaining  the  corpuscles  has  been  described  in  the  early 
part  of  this  work. 

In  the  complement-fixation  test,  there  are  also  used,  besides  the 
hemolytic  system,  the  serum  of  the  horse  to  be  examined  and  antigen. 

METHOD   OF   OBTAINING  SERUM   OF  ANIMALS   TO   BE   TESTED. 

The  blood  is  drawn  from  the  jugular  vein  of  the  suspected  horse 
after  a  small  area  over  the  jugular  has  been  clipped  and  disinfected 
with  alcohol.  The  vein  is  dilated  by  pressure  on  the  lower  part  of 
the  neck,  and  the  blood  is  drawn  from  the  animal  by  the  insertion  of 
a  trocar  and  cannula.  It  is  recommended  that  the  blood  should  be 
collected  in  uniform-sized  tubes  or  bottles,  which  should  be  sterilized 
before  using.    A  sufficient  quantity  of  blood  for  testing  purposes 


OBTAINlNa  SERtJM   OP   SUSPECTED   HORSE.  19 

would  be  50  to  100  c.  c,  and  after  allowing  the  blood  serum  in  the 
tube  to  separate  from  the  clot  in  a  cool,  dark  place  it  is  ready  to  be 
used  for  the  test.  If  it  is  desired  to  forward  the  blood  to  a  laboratory 
the  tubes  may  be  packed  into  separate  containers  or  collectively  in  a 
box.  Every  tube  should  be  labeled,  and  the  number  of  the  horse 
corresponding  with  the  record  number  should  be  designated  on  the 
label.  It  is  not  absolutely  essential  to  have  clear  serum,  as  in  repeated 
tests  carried  out  in  this  laboratory  it  was  found  that  blood  forwarded 
from  Michigan  to  Washington  gave  satisfactory  reactions,  although 
the  serum  was  badly  discolored  as  a  result  of  disintegration  of  the 
blood  corpuscles.  If  it  is  desired  to  preserve  the  serum,  or  if  from 
some  cause  a  test  can  not  be  applied  during  the  first  few  days  after 
the  blood  has  been  drawn,  it  should  be  preserved  with  a  0.5  per  cent 
solution  of  carbolic  acid.    This  percentage  is  best  obtained  by  adding 

1  part  of  a  5  per  cent  carbolic-acid  solution  to  9  parts  of  the  serum  to 
be  preserved.  Such  carbolized  serum  will  respond  to  this  test  after 
several  months. 

In  cases  where  the  mallein  test  has  been  used  the  blood  of  sus- 
pected horses  to  be  examined  for  glanders  by  complement  fixation 
should  not  be  taken  until  from  7  to  10  days  have  elapsed  after  the 
last  mallein  test.  This  is  necessary  because  of  the  possibility  that 
the  injected  mallein  may  have  exerted  a  stimulating  effect  on  the  cells 
with  the  production  of  immune  bodies,  and  if  serum  is  then  taken 
for  the  test  the  results  may  lead  to  a  faulty  diagnosis.  However,  in 
suspected  cases  of  glanders  the  blood  of  the  animals  may  be  drawn 
and  forwarded  for  examination  to  a  laboratory  where  the  serum 
diagnosis  of  glanders  is  practiced,  without  the  necessity  of  the  appli- 
cation of  the  mallein  test. 

INACTIVATING    THE    SERUM. 

Of  the  horse  serum  to  be  examined  about  2  c.  c.  is  drawn  off  and 
placed  in  a  suitable  tube  or  bottle  in  order  to  subject  it  to  a  tempera- 
ture of  58°  C.  in  a  water  bath  for  one-half  hour.  This  constitutes  the 
inactivation  of  the  serum ;  that  is,  the  complement  which  is  present  in 
the  serum  is  destroyed  by  this  heating.  Such  inactivated  serum  is 
ready  for  use  in  the  testing,  but  should  be  used  only  on  the  day  of  its 
inactivation.    In  case  it  becomes  necessary  to  repeat  the  test,  another 

2  c.  c.  of  the  sample  should  be  inactivated. 

The  method  of  inactivation  referred  to  above  applies  only  to 
horses.  Miessner  and  Trapp  found  that  serum  of  mules  inactivated 
at  58°  C.  and  59°  C.  does  not  in  all  cases  give  satisfactory  results, 
as  in  many  instances  even  the  normal  serum  of  these  animals  checks 
hemolysis.  The  numerous  tests  which  were  carried  out  in  this  labor- 
atory with  serum  of  horses  and  mules  proved  that  while  the  inactiva- 
tion of  the  horse  serum  at  58°  C.  and  59°  C.  always  gave  satisfactory 
results,  the  tests  made  with  mule  serum  under  the  same  conditions 
were  far  from  uniform. 


20  DIAGNOSIS   OF   GLANDERS. 

Accordingly,  it  was  deemed  advisable  to  inactivate  sera  from  mules 
at  a  higher  temperature  as  well  as  by  carbolization.  Normal  mule 
serum  was  subjected  to  various  degrees  of  temperature  ranging  from 
58°  C.  to  62°  C.  for  three- fourths  to  one  hour,  and  similar  samples 
of  serum  were  carbolized  and  inactivated  at  the  same  temperature 
as  the  noncarbolized  serum.  The  results  showed  that  carbolized  mule 
serum  inactivated  at  60°  C.  gives  the  most  uniform  results  in  the  com- 
plement-fixation test.  The  same  serum  if  not  carbolized  showed 
checking  of  hemolysis  at  an  inactivation  recommended  for  horse 
serum.  Inactivations  at  a  higher  degree  of  temperature  are  not 
advisable,  inasmuch  as  at  62°  C.  a  considerable  percentage  of  sera 
will  coagulate.  Therefore,  in  testing  mule  sera  a  carbolization  with 
0.5  per  cent  carbolic  acid  followed  by  an  inactivation  at  60°  C.  for 
one  hour  is  advisable. 

The  antihemolytic  action  of  the  normal  sera  of  mules  is  probably 
due  to  the  presence  of  substances  in  the  blood  which  exert  a  checking 
action  on  hemolysis,  as  glycogen,  pepton,  albumose.  These  undeter- 
mined substances  act  by  using  up  the  complement,  having  a  greater 
affinity  toward  it  than  the  hemolytic  amboceptor  has. 

PREPARATION  OF  THE  ANTIGEN  (GLANDERS  BACILLI  EXTRACT). 

The  antigen  represents  a  shake  extract  of  glanders  bacilli  in  salt 
solution.  It  is  prepared  as  follows :  From  a  stock  culture  of  glanders 
bacilli  subcultures  are  made  on  2  per  cent  acid  glycerin  agar  media* 
It  is  preferable  to  use  Kolle  flasks  instead  of  tubes  for  the  cultures, 
inasmuch  as  in  such  flasks  the  surface  of  the  media  is  much  larger 
and  a  greater  quantity  of  bacilli  can  be  obtained  from  them.  After 
inoculating  the  media  with  glanders  bacilli  the  flasks  are  placed  in 
the  incubator,  and  after  24  hours  the  condensation  water  in  the  cul- 
tures is  allowed  to  run  over  the  surface  of  the  media.  After  another 
24  or  48  hours  in  the  incubator  the  surface  of  the  media  contains 
usually  a  luxuriant  growth  of  glanders  bacilli,  and  it  is  then  ready 
for  washing.  To  each  flask  20  to  40  c.  c.  of  salt  solution  is  added.  If 
the  cultures  have  been  grown  in  ordinary  test  tubes  they  are  washed 
off  with  5  to  15  c.  c.  of  salt  solution. 

After  the  entire  growth  is  washed  off  the  surface  of  the  medium 
the  fluid  is  poured  into  sterile  flasks  and  then  heated  to  60°  C.  for 
four  hours  in  order  to  kill  the  glanders  bacilli.  After  the  heating 
of  the  bacilli  the  flasks  are  placed  in  a  shaking  apparatus  and  shaken 
for  four  days. 

After  removal  of  the  flasks  the  extract  is  placed  in  centrifuge  tubes 
and  centrifugalized  for  two  hours  in  a  centrifuge  at  a  speed  of  2,500 
to  3,000  revolutions  per  minute.  Then  to  the  clear  liquid,  which  has 
been  drawn  off  into  suitable  bottles,  10  per  cent  of  a  5  per  cent 
carbolic-acid  solution  is  added,  and  the  bottles  are  corked.    This  rep- 


BuL.   136,  Bureau  of  Animal  Industry,  U.S.  Dept.  of  Aoriculture. 


Plate   IV. 


TITRATION   OF   GLANDERS  BACIUL.I   EXTRACT.  21 

resents  the  extract  or  antigen  to  be  used  for  the  tests  in  a  dihition 
which  is  established  by  titration.  The  extract  usually  keeps  for  two 
or  three  months,  or  sometimes  even  longer  if  kept  in  a  dark,  cool 
place.  . 

TITRATION  OF  THE  EXTRACT. 

The  titration  of  the  extract  is  carried  out  in  order  to  establish  the 
quantity  of  the  extract  which  no  longer  prevents  hemolysis.  First, 
dilutions  with  salt  solution  are  made  from  the  extract  in  proportions 
of  1  to  20,  30,  40,  50,  and  so  on  up  to  200.  These  dilutions  are  made 
from  a  basic  dilution  of  1  to  10,  as  can  be  seen  from  Table  3.  The 
titration  proper  is  carried  out  as  follows:  Nine  test  tubes  are  used 
for  the  titration  and  three  for  controls.  To  each  tube  1  c.  c.  of  salt 
solution  is  added,  then  1  c.  c.  of  complement  of  the  determined  small- 
est quantity  established  according  to  the  preliminary  test  (see  Table 
2).  This  is  followed  by  the  extract,  each  tube  receiving  1  c.  c.  of  the 
different  dilutions  prepared.  Thus,  in  the  first  tube  1  c.  c.  of  the 
dilution  of  1  to  10  is  added ;  in  the  second,  1  c.  c.  of  the  dilution  of 
1  to  20;  in  the  third,  1  c.  c.  of  the  dilution  of  1  to  30,  and  so  on. 
Then  the  rack  is  placed  in  the  incubator  for  one  hour.  After  it  is 
taken  out,  1  c.  c.  of  a  double  quantity  of  the  previously  titrated  ambo- 
ceptor is  added  to  each  tube,  and  finally  1  c.  c.  of  a  5  per  cent  sus- 
pension of  washed  sheep  blood  corpuscles.  Of  the  three  control 
tubes,  the  first  serves  for  a  control  to  show  that  the  complement  alone 
(without  the  amboceptor)  does  not  produce  hemolysis,  the  second  to 
show  that  the  amboceptor  alone  does  not  produce  hemolysis,  and  the 
third  that  the  salt  solution  alone  does  not  produce  hemolysis.  Thus, 
in  the  first  control  tube  3  c.  c.  of  salt  solution,  1  c.  c.  of  complement, 
and  1  c.  c.  of  blood  corpuscles  are  added.  The  second  tube  contains 
3  c.  c.  of  salt  solution,  1  c.  c.  of  amboceptor,  and  1  c.  c.  of  blood  cor- 
puscles, while  the  third  contains  4  c.  c.  of  salt  solution  and  1  c.  c.  of 
blood  corpuscles.  This  titration  is  similar  to  the  titration  of  the 
hemolytic  amboceptor. 

After  shaking  the  test  tubes,  which  should  invariably  follow  all 
these  additions,  the  rack  is  placed  in  the  incubator  for  two  hours. 
Then  the  results  are  read.  The  tube  in  which  hemolysis  is  no  longer 
prevented  represents  the  titre  of  the  extract.  (See  PI.  IV.)  In  the 
glanders  test,  however,  one-half  of  that  quantity  is  used.  Thus,  if 
the  titration  should  prove  that  the  first  tube  which  does  not  prevent 
hemolysis  contains  a  dilution  of  1  to  50,  then  a  dilution  of  1  to  100 
of  the  extract  is  used  for  the  glanders  tests. 

In  Table  3  the  titration  of  the  glanders  bacilli  extract  is  given  in 
consecutive  order,  and  this  may  be  followed  in  a  similar  manner  as 
with  the  other  tables.  In  this  table  the  substances  to  be  used  are 
given  consecutively,  and  likewise  the  exact  quantities  are  indicated, 


DIAGNOSIS   OF   GLANDERS. 
Table  3. — Titration  of  glanders  bacilli  extract. 


Tube. 

NaCl 
solution.! 

Comple- 
ment.' 

Extract.' 

Ambo- 
ceptor.< 

Blood 

cor- 

puscles.5 

Remarks. 

• 

1 
2 
3 
4 
5 
6 
7 
8 
9 

c.c. 

c.c. 
1 

1 
1 
1 
1 
1 
1 
1 
1 

1  c.  c.  of— 
1:10... 
1:20... 
1:30... 
1:40... 
1:50... 
1:60... 
1:80... 
1  :100.. 
1  :  200. . 

1 
1 

3 
1 

c.  c. 

c.  c. 

1 
1 

1 
1 
1 
1 
1 
1 
1 

One-half  of  the  quantity  of  the  ex- 
tract which  no  longer  prevents 
hemolysis  is  the  proper  value  to 

■  be  used  in  future  final  testing. 
Thus,  if  it  should  show  by  this 
test  to  be  1  to  50,  then  a  dilution 
of  1  to  100  of  the  extract  is  used. 

10 

3 
3 
4 

1 

1 
1 
1 

Complement  control  (no  hemolysis 

11 

1 

should  occur). 
Amboceptor  control  (no  hemolysis 

12 

should  occur). 
Salt  solution  control  (no  hemolysis 

should  occur). 

>  0.85  per  cent  NaCl  solution. 

'  The  determined  smallest  quantity  established  according  to  the  preliminary  test. 
'  A  basic  dilution  is  made  from  the  extract  in  proportions  of  1  to  10,  20,  30,  etc.,  up  to  200.    From  these 
basic  dilutions  the  third  column  is  made  as  indicated  below. 
*  Double  the  quantity  previouslv  determined  by  titration. 
5  5  per  cent  suspension  of  sheep-blood  corpuscles  washed  in  salt  solution. 

The  basic  dilutions  of  the  glanders  bacilli  extract  are  made  as 
follows : 

A  dilution  of— 

1  to  20  is  made  by  adding  1  c. 
1  to  .30  is  made  by  adding  1  c. 
1  to  40  is  made  by  adding  1  c. 
1  to  50  is  made  by  adding  1  c. 
1  to  60  is  made  by  adding  1  c. 
1  to  80  is  made  by  adding  1  c. 
1  to  100  is  made  by  adding  1  c. 
1  to  200  is  made  by  adding  1  c. 


c. 

of  1  to 

10  dilution  to  1  e. 

c. 

NaOl  solution. 

c. 

of  1  to 

10  dilution  to  2  c. 

c. 

NaCl  solution. 

c. 

of  1  to 

10  dilution  to  3  c. 

c. 

NaCl  solution. 

c. 

of  Ito 

10  dilution  to  4  c. 

c. 

NaCl  solution. 

c. 

of  1  to 

10  dilution  to  5  c. 

c. 

NaCl  solution. 

c. 

of  Ito 

10  dilution  to  7  c. 

c. 

NaCl  solution. 

c. 

of  Ito 

10  dilution  to  9  c. 

c. 

NaCl  solution. 

c 

of  1  to  100  dilution  to  1  c. 

c 

NaCl  solution. 

DIAGNOSIS   OF   GLANDERS.  23 

THE   COMPLEMENT-FIXATION   TEST. 

As  previously  stated,  the  serum  of  glandered  horses  contains, 
immune  bodies  which  develop  during  the  course  of  the  disease.  The 
presence  of  these  immune  bodies  in  the  serum  is  utilized  in  the  devel- 
opment of  the  test.  Naturally,  the  amount  of  these  immune  bodies 
in  the  affected  horses  is  not  uniform  and  undoubtedly  depends  on  the 
extent  of  the  infection  present ;  therefore  it  is  advisable  to  use  in  the 
test  such  quantities  of  the  serum  as  will  prove  sufficient  for  the  reac- 
tion to  take  place. 

The  necessary  quantity  for  the  test  has  accordingly  been  established, 
through  the  painstaking  experiments  of  Shiitz  and  Schubert,  as  0.2 
and  0.1  c.  c,  placed  in  two  different  tubes.  In  some  instances  the  tube 
containing  0.2  c.  c.  of  the  serum  may  show  a  fixation  of  the  comple- 
ment, while  the  tube  containing  0.1  c.  c.  of  the  same  serum  may  show 
only  a  partial  fixation  or  hemolysis.  In  the  great  majority  of  cases, 
however,  the  fixation  is  usually  manifested  in  both  tubes. 

The  substances  which  are  used  in  the  fixation  test  have  already  been 
described  in  detail,  their  strength  has  been  established  by  the  titration 
of  these  different  substances,  and  it  need  only  be  mentioned  in  passing 
that  the  best  results  will  be  obtained  when  the  smallest  quantities  of 
substances  permitted  by  the  titration  are  used  for  the  test;  that  is, 
the  substances  should  have  a  high  titre.  If,  for  instance,  the  glanders 
extract  should  prove  of  a  low  titre,  the  albumins  which  are  contained 
therein  may  have  an  influencing  effect  upon  the  outcome  of  the  test, 
inasmuch  as  it  is  known  that  albumins  or  cell  extracts,  if  present  in 
large  quantities,  will  fix  complements.  Therefore  it  is  always  advis- 
able to  use  glanders  extract  of  a  high  titre;  the  quantity  to  be  used 
should  not  be  much  above  0.01  c.  c. 

Besides  the  centrifugal  machine  and  an  ordinary  shaking  appara- 
tus, only  glassware  is  needed  for  the  test.  This  consists  principally 
of  pipettes  of  various  sizes,  and  there  should  always  be  a  sufficient 
number  on  hand  during  the  performance  of  the  test.  Those  best 
adapted  for  the  work  are  1  c.  c.  pipettes,  graduated  into  hundredths, 
and  10  c.  c.  pipettes,  graduated  into  tenths.  In  addition,  several 
pipettes  of  various  sizes  will  be  found  useful  for  measuring  the 
various  substances  to  be  diluted.  Uniform-sized  test  tubes  are 
advised  and  they  should  fit  into  test-tube  racks.  All  the  glassware 
should  be  cleaned  and  dry-sterilized  before  using.  Only  such  a  num- 
ber of  tests  as  can  be  conveniently  handled  should  be  undertaken  on 
a  single  day.  Finally,  the  incubator  should  contain  sufficient  space 
to  hold  the  necessary  number  of  racks  during  the  test.  With  a  good- 
sized  incubator  and  the  above-mentioned  apparatus  any  State  bacteri- 
ological laboratory  could  meet  the  demand  for  diagnoses  from  all 


24  DIAGNOSIS   OF   GLANDERS. 

the  veterinarians  within  its  borders,  as  from  80  to  100  tests  may 
readily  be  made  by  this  method  in  one  day. 

APPLICATION  OF  THE  TEST. 

Before  undertaking  the  tests  it  is  advisable  to  prepare  the  dilu- 
tions which  will  be  used  for  the  testing.  The  quantities  of  dilutions 
can  be  determined  quite  accurately,  in  accordance  with  the  number  of 
tests  to  be  made.  Thus  the  necessary  quantity  of  a  suspension  of 
blood  corpuscles  is  prepared,  as  well  as  the  dilution  of  the  necessary 
quantity  of  the  amboceptor,  complement,  and  glanders  extract.  The 
preparation  of  these  dilutions  is  best  accomplished  in  Erlenmeyer 
flasks,  and  the  dilutions  prepared  should  not  be  kept  over  one  day. 

Four  test  tubes  are  used  in  testing  the  serum  of  each  horse.  For 
convenience  in  reading  the  results  they  are  placed  in  a  test-tube  rack. 
The  first  test  tube  is  marked  with  the  number  of  the  animal  corre- 
sponding with  that  on  the  record  for  identification.  Thus,  for  in- 
stance, should  it  be  necessary  to  examine  20  horses,  80  tubes  would 
be  required  for  the  tests.  The  first  and  third  tubes  are  intended  for 
the  test  proper,  while  the  second  and  fourth  tubes  serve  as  controls 
for  the  horse  serum  to  be  examined.  All  the  test  tubes  are  first  filled 
with  the  necessary  quantity  of  salt  solution,  which  will  make  up  with 
the  other  substances  used  the  required  5  c.  c.  Thus  the  first  and  third 
tubes  each  contain  1  c.  c.  of  salt  solution,  while  the  second  and  fourth 
tubes  contain  2  c.  c.  each.  Then  the  suspected  horse  serum  to  be  ex- 
amined, which  has  been  previously  inactivated  by  heating  for  one-half 
hour  at  58°  C,  is  added.  In  the  first  and  second  tubes  0.1  c.  c.  and  in 
the  third  and  fourth  tubes  0.2  c.  c.  of  this  inactivated  serum  is  added. 
This  is  followed  by  the  addition  of  the  glanders  bacilli  extract,  which, 
however,  is  only  introduced  into  the  first  and  third  tubes,  1  c.  c.  of 
the  established  dilution  being  used.  The  second  and  fourth  tubes, 
therefore,  do  not  contain  glanders  bacilli  extract,  as  these  tubes  serve 
only  as  controls  to  show  that  the  horse  serum  alone  will  not  influence 
hemolysis.  Thus  in  every  test  the  amount  of  serum  used  in  the  tests 
proper  (first  and  third  tubes)  is  controlled  by  an  equal  amount  of 
serum  (second  and  fourth  tubes). 

After  the  addition  of  the  antigen  (glanders  extract),  1  c.  c.  of  the 
complement  is  added  to  every  tube  in  a  dilution  which  has  been 
established  by  the  preliminary  tests.     (See  Table  2.) 

At  this  stage  of  the  testing  each  tube  contains  3  c.  c.  of  fluid.  The 
rack  containing  the  tubes  is  now  shaken  and  placed  in  the  incubator 
for  one  hour.  This  incubation  is  carried  out  in  order  to  allow  the 
anchoring  of  the  antigen  and  the  glanders  immune  body,  in  which 
the  complement  will  become  firmly  fixed.    Naturally,  the  anchoring 


THE   COMPLEMENT -FIXATION   TEST.  25 

would  only  take  place  provided  the  serum  contains  the  glanders  im- 
mune bodies;  that  is,  when  the  horse  is  infected  with  glanders.  (See 
PI.  I,  D.)  On  the  other  hand,  in  the  absence  of  a  glanders  infection 
the  serum  does  not  contain  immune  bodies,  and  therefore  the  antigen 
as  well  as  the  complement  will  remain  free  in  the  test  tubes.  (See 
PI.  I,  C.) 

After  the  time  required  for  the  incubation  has  elapsed  the  rack  is 
removed  and  the  other  substances  of  the  hemolytic  system  are 
added,  namely,  the  hemolytic  amboceptor  and  the  blood-corpuscles 
suspension. 

To  each  test  tube  is  added  1  c.  c.  of  the  previously  titrated  rabbit 
serum  (hemolytic  amboceptor),  and  finally  1  c.  c.  of  a  5  per  cent  sus- 
pension of  sheep  blood  corpuscles  which  have  been  previously  washed 
with  salt  solution.  The  tubes,  either  individually,  or  collectively  in 
the  rack,  should  then  be  well  shaken  by  hand,  in  order  to  mix  the 
fluids  thoroughly  and  to  wash  down  any  flecks  of  the  solutions  that 
might  have  adhered  to  the  sides  of  the  tubes. 

This  practically  concludes  the  test,  and  it  is  now  only  necessary  to 
incubate  the  tubes  for  10  hours,  when  the  results  may  be  read. 

CONTROLS. 

During  the  application  of  all  tests  control  tubes  are  also  used  at 
the  same  time  for  comparative  purposes.  Four  tubes  are  required 
for  the  control  of  the  hemolytic  system  and  two  for  the  control  of 
the  glanders  extract.  The  control  of  the  hemolytic  system  is  es- 
tablished by  using  in  the  first  tube  all  substances  constituting  the 
hemolytic  system,  namely,  the  hemolytic  amboceptor,  the  comple- 
ment, and  the  suspension  of  blood  corpuscles.  This  tube  after  the 
conclusion  of  the  test  should  show  a  complete  hemolysis.  In  the 
second  tube  the  hemolytic  amboceptor  is  controlled  without  the  com- 
plement, to  prove  that  it  does  not  produce  hemolysis.  The  third  tube 
serves  as  a  control  of  the  complement  without  the  hemolytic  system, 
also  to  prove  that  it  does  not  produce  hemolysis.  The  fourth  tube  is 
to  show  that  the  salt  solution  has  no  hemolytic  action  on  the  blood 
corpuscles.  The  quantities  of  the  different  substances  used  in  the 
controls  are  the  same  as  used  in  the  regular  tests  for  glanders.  It  is 
necessary  in  the  control  tubes,  as  well  as  in  all  other  tubes,  to  make 
up  the  quantity  to  5  c.  c.  with  salt  solution.  Thus,  in  the  tube  which 
serves  for  the  control  of  the  hemolytic  system,  2  c.  c.  of  salt  solution 
is  added,  while  in  the  control  of  the  hemolytic  amboceptor  as  well  as 
in  the  control  of  the  complement  3  c.  c,  and  in  the  salt-solution  con- 
trol 4  c.  c,  are  added.    The  substances  added  to  the  control  tubes  are 


26  DIAGNOSIS  OF   GLANDERS. 

used  in  the  same  order  as  given  in  the  test,  and  the  incubation  is  also 
followed  at  the  same  time. 

As  above  stated,  two  test  tubes  are  used  for  the  control  of  the  anti- 
gen (glanders  bacilli  extract),  in  addition  to  the  four  used  for  the 
control  of  the  hemolytic  system.  These  two  tubes  contain  the  hemo- 
lytic system,  and  to  the  first  tube  1  c.  c.  of  the  extract  used  in  the 
glanders  test  is  added,  while  to  the  second  tube  is  added  2  c.  c.  of  the 
extract.  The  controls  of  the  extract  serve  to  prove  that  the  extract 
used  in  the  tests  does  not  cause  a  fixation  of  the  complement,  and  that 
even  double  the  quantity  of  the  extract  which  has  been  used  in  the 
glanders  test  does  not  fix  the  complement.  In  the  extract  controls 
we  have  in  the  first  1  c.  c.  of  salt  solution,  while  to  the  second  tube 
no  salt  solution  is  added,  inasmuch  as  the  substances  in  this  control 
make  up  the  necessary  5  c.  c.  In  the  first  tube,  besides  the  1  c.  c.  of 
salt  solution,  1  c.  c.  of  the  extract  is  added,  which  is  followed  in  con- 
secutive order  with  the  substances  of  the  hemolytic  system,  in  quan- 
tities and  dilutions  used  during  the  test  proper,  which  have  been 
previously  indicated. 

The  results  in  the  control  tubes  after  the  conclusion  of  the  tests 
should  show  the  following: 

All  the  control  tubes  which  contain,  besides  the  hemolytic  system, 
only  serum  of  the  suspected  horse — that  is,  the  second  and  fourth 
tubes — should  show  a  complete  or  almost  complete  hemolysis.  There 
should  be  no  deposit  of  blood  corpuscles  on  the  bottom  of  these  tubes, 
or  only  a  very  small  quantity.  The  fluid  should  have  the  charac- 
teristic (laked)  wine-red  color. 

In  the  control  tubes  of  the  extract  there  should  also  be  a  complete 
or  almost  complete  hemolysis.  The  same  should  be  present  in  the 
control  of  the  hemolytic  system ;  it  should  show  a  complete  hemolysis. 
In  the  control  tubes  of  the  complement  the  hemolytic  ambocepter, 
and  the  salt  solution,  there  should  be  no  hemolysis  whatever ;  that  is, 
the  blood  corpulscles  should  be  settled  in  the  bottom  of  the  tube,  and 
the  liquid  above  them  should  be  water-clear. 

In  Table  4  the  final  test  for  glanderg  is  given  in  the  order  in  which 
it  should  be  undertaken.  The  number  of  test  tubes  to  be  used  is 
indicated,  and  also  the  number  of  controls  to  be  made.  The  sub- 
stances and  quantities,  as  well  as  the  dilutions,  are  also  indicated  in 
the  explanatory  remarks  of  the  table.  As  already  stated,  six  controls 
are  sufficient  for  any  number  of  tests  undertaken  on  one  day,  if  the 
same  substances  are  used  for  all  the  tests.  In  making  the  comple- 
ment-fixation test  it  is  always  advisable  to  control  the  results  further 
by  using  serum  from  an  established  case  of  glanders  in  one  series  of 
four  tubes,  and  from  a  healthy  horse  in  another  similar  series,  for 
comparative  purposes. 


BuL.  136,  Bureau  of  Anim*l  Industry.  U.  S.  Dept.  of  Aqriculture. 


Plate  V. 


THE   COMPLEMENT-FIXATION    TEST. 
Table  4. — The  final  test  for  glanders. 


27 


Tube.L„S„, 

Suspected  |  Glanders 

horse      '    bacilli 

serum.'    1  extract.' 

1 

Comple- 
ment.* 

Ambo- 
ceptor.* 

Blood 
cor- 
puscles.' 

Remarks. 

1 
2 

e.c. 
1 

2 

1 
2 

c.  c. 

ai 
.1 

•2 

.2 

c.  c. 

1 

e.  c. 

i 

03 

3 
o 
d 

_d 

§ 

c.  c. 

c.  c. 

Test  tube  for  the  dose 
0.1  c.  c.  of  suspected 
senun. 

Serum   control    for    the 

3 

4 

1 

dose  0.1  0.  c.  of  sus- 
pected serum. 

Test  tube  for  the  dose 
0.2  c.  c.  of  suspected 
serum. 

Serum   control    for    the 

dose  0.2  c.  c.  of  sus- 
pected sdrum. 
Control  for  the  quantity 
of  extract  used  (hemo- 
lysis). 

S 

1 

I 
2 

6 

quantity  of  extract, 
for  greater  accuracy 
(hemolysis). 

Control  of  the  hemolytic 
system  (hemolysis). 

Control  of  the  comple- 
ment (no  hemolysis). 

Control  of  amboceptor 
(no  hemolysis). 

Control  of  salt  solution 

7 

2 
3 
3 

4 

8 

9 

10 

(no  hemolysis). 

1 0.85  per  cent  NaCl  solution. 

'Suspected  horse  serum  to  be  inactivated  for  30  minutes  at  58°  C.  in  water  bath,  in  order  to  destroy  the 
complement  which  is  present  in  the  serum  of  every  horse. 
» One-half  of  the  quantity  which  does  not  prevent  hemolysis  and  established  by  titration.    (See  Table  3.) 
<  The  determined  smallest  quantity  established  according  to  the  preliminary  test.    (See  Table  2.) 
6  Double  the  quantity  previously  determined  by  titration.    (See  Table  1.) 
*5  per  cent  suspension  of  washed  sheep-blood  corpuscles. 


INTERPRETING    RESULTS    OF    TESTS. 

The  results  of  the  tests  are  manifested  in  most  instances  by  a  dis- 
tinct reaction  which  takes  place  in  the  test  tubes.  The  indication  of 
the  results  is  only  to  be  sought  in  the  tubes  numbered  1  and  3,  which 
represent  the  actual  tests  for  the  presence  or  absence  of  glanders,  inas- 
much as  these  tubes  contain  all  substances  required  in  the  tests. 

We  may  thus  obtain  in  these  tubes  either  complete  hemolysis,  in- 
complete hemolysis,  or  no  hemolysis  whatsoever.  The  fixation  of  the 
complement  is  manifested  by  the  absence  of  hemolysis,  and  therefore 
we  have  in  the  first  and  third  tubes  a  settling  of  the  blood  corpuscles 
with  the  watery  clear  fluid  above.  Such  a  result  indicates  without 
doubt  the  presence  of  glanders.  On  the  other  hand,  if  the  first  and 
third  tubes  show  complete  hemolysis,  the  absence  of  glanders  is 
thereby  indicated.  In  the  presence  of  glanders  a  fixation  of  the 
complement  takes  place,  as  a  result  of  anchoring  to  the  immune  bodies 
and  antigen  (see  PI.  I,  D),  while  in  the  absence  of  glanders,  there 
being  no  immune  bodies  present,  the  complement  is  used  up  in  the 
phenomenon  of  hemolysis  (see  PI.  I,  C). 

Then,  again,  we  may  have  cases  in  which  the  fixation  of  the  com- 
plement is  incomplete ;  that  is,  there  is  a  settling  of  corpuscles  in  the 
bottom  of  the  test  tube,  but  the  fluid  shows  traces  of  hemolysis.     It 


28  DIAGNOSIS   OF   GLANDERS. 

does  not  show  the  characteristic  saturated  color  of  hemolysis,  but 
only  a  tingeing  with  the  hemoglobin.  This  is  termed  an  almost  com- 
plete fixation,  and  also  indicates  the  presence  of  glanders.  The  pres- 
ence of  the  characteristic  color  in  the  fluid  and  a  very  slight  deposit 
of  corpuscles  on  the  bottom  should  not  be  taken  as  an  indication  of  an 
infection,  as  such  a  condition  may  be  brought  on  by  various  causes,  and 
particularly  so  by  the  presence  of  nonspecific  substances  in  the  serum 
of  the  horse,  which  may  cause  a  very  slight  checking  of  the  hemolysis. 
But  all  cases  where  the  results  show  a  fixation  of  the  complement  (no 
hemolysis)  or  almost  complete  fixation  (slight  tingeing  of  the  fluid 
above  the  settled  blood  corpuscles)  indicate  the  presence  of  glanders. 

As  a  result  of  the  different  quantities  of  horse  serum  used  in  the 
two  pairs  of  test  tubes,  it  will  be  found  occasionally  that  the  test 
tube  containing  the  larger  quantity  of  serum  (0.2  c.  c. — third  tube) 
shows  a  fixation  of  the  complement,  while  the  tube  containing  the 
smaller  quantity  of  serum  (0.1  c  c. — first  tube)  shows  a  partial  fixa- 
tion of  the  complement.  This  also  can  be  considered  as  a  glanderous 
infection,  as  it  shows  that  there  are  present  immune  bodies,  but  not 
to  such  an  extent  as  to  produce  the  fixation  of  the  complement  in  the 
tube  where  the  smaller  quantity  of  the  suspected  horse  serum  has 
been  used.  In  most  instances  of  glanderous  infections,  however,  it 
will  be  observed  that  the  fixation  of  the  complement  is  uniform  and 
almost  complete  in  both  tubes. 

The  reading  should  be  made  after  the  results  in  the  test  tube  have 
been  clearly  established,  and  one  should  not  be  too  hasty  in  drawing 
conclusions  before  the  reaction  in  the  test  tubes  is  clear  enough  to 
establish  the  results  of  the  test. 

In  the  illustrations  in  Plate  V  the  results  of  the  glanders  tests  are 
indicated  by  the  presence  of  a  positive  reaction.  In  tubes  1  and  3  a 
complete  fixation  of  the  complement  has  taken  place,  there  being  no 
hemolysis,  while  tubes  2  and  4  represent  the  controls  for  the  horse 
serum  (no  glanders  extract  being  added),  these  tubes  showing  hemo- 
lysis. In  the  upper  row  of  the  rack  the  controls  are  indicated,  and  they 
show  the  results  as  they  should  be  obtained  in  the  course  of  testing. 

Should  the  horse  prove  glandered  on  the  above  test,  the  test  may  be 
repeated,  and  then  serum  quantities  are  used  as  follows:  0.1,  0.05, 
0.03,  0.02,  and  0.01  c.  c.  One  control  is  made  of  0.1  c.  c.  as  in  tube  2 
of  the  test.  By  this  method  the  smallest  quantity  of  serum  is  estab- 
lished which  will  divert  the  complement,  but  it  is  not  necessary  as 
routine  practice. 

Our  own  experience,  as  well  as  the  work  of  Schiitz  and  Schubert, 
to  whom  we  wish  to  extend  our  acknowledgments,  shows  that  the  results 
of  the  complement-fixation  test  should  be  interpreted  as  follows : 

1.  Horses  in  which  the  serum  produces  a  complete  fixation  of  the 
complement  in  the  quantities  of  0.1  c.  c.  and  0,2  c.  c.  should  be  con- 
sidered as  glandered. 


RESULTS  OF  PRACTICAL,  TESTS.  29 

2.  Horses  in  which  the  serum  gives  a  complete  fixation  in  the 
quantity  of  0.2  c.  c.  and  an  incomplete  fixation  in  the  quantity  of  0.1 
c.  c.  should  likewise  be  considered  as  glandered, 

3.  Horses  in  which  the  serum  produces  an  incomplete  fixation  of  the 
complement  in  the  quantities  of  0.1  c.  c.  and  0.2  c.  c.  should  also  be 
considered  as  glandered. 

I  4.  Horses  in  which  the  serum  shows  no  fixation  of  the  complement 
in  either  tube  should  be  considered  free  of  glanders. 

In  order  to  reduce  the  possibility  of  error  to  a  minimum  the 
agglutination  test  may  be  applied  to  the  latter  cases,  and  if  this 
shows  a  value  of  1  to  1,000  or  over,  the  animal  should  be  considered 
as  glandered.     However,  such  cases  are  extremely  rare. 

The  agglutination  test  may  be  undertaken  with  the  complement- 
fixation  test  without  a  great  deal  of  difficulty,  especially  if  the 
agglutination  test  is  carried  out  in  accordance  with  the  method  used 
at  the  present  time  in  Germany,  by  which  the  results  of  the  test  can 
be  obtained  in  about  two  hours.  This  consists  of  a  modification  of 
the  agglutination  test,  in  which  by  centrifugalization  the  agglutina- 
tion is  hastened  in  the  test  tubes  and  the  results  can  be  read  after 
the  tubes  have  been  placed  in  the  incubator  for  two  hours. 

The  technique  of  this  method  of  agglutination  testing  will  not  be 
explained  here,  but  it  is  planned  to  describe  it  in  a  later  publication 
on  this  subject. 

CONTROLLING  GLANDERS  IN  AN  INFECTED  STABLE. 

When  glanders  is  discovered  or  suspected  among  the  horses  in  a 
stable,  the  blood  of  all  the  horses  in  the  infected  stable  should  be 
drawn  and  tested  in  the  manner  previously  described.  All  animals 
whose  serum  shows  a  complement  fixation  should  be  destroyed  with- 
out further  consideration.  After  the  animals  have  been  killed  the 
stable  should  be  thoroughly  cleaned  and  disinfected.  The  animals 
which  gave  no  reaction  on  the  first  test  should  be  retested  after  three 
weeks,  and  should  there  be  no  indication  of  the  disease  in  the  second 
test  the  stable  may  be  considered  free  from  the  infection.  On  the 
other  hand,  if  on  the  second  test  one  or  more  animals  should  give  a 
reaction,  the  infected  animals  should  be  destroyed  and  all  the  re- 
maining horses  should  again  be  subjected  to  another  retest  after 
three  weeks.  Not  until  the  last  test  proves  that  no  animal  is  affected 
can  the  stable  be  considered  as  free  from  the  infection. 

RESULTS   OF   PRACTICAL   TESTS   WITH    COMPLEMENT    FIXATION. 

In  order  to  ascertain  how  long  a  time  after  infection  with  glan- 
ders elapses  before  the  presence  of  the  disease  could  be  detected  by 
the  complement-fixation  test,  a  horse  was  infected  with  glanders  at 
the  Bureau  of  Animal  Industry  Experiment  Station  by  taking  a 


80  DIAGNOSIS   OF   GLANDERS. 

loopful  of  glanders  culture  and  rubbing  it  up  and  down  the  Schnei- 
derian  membrane.  Twenty-four  hours  after  the  infection  the  first 
sample  of  blood  was  taken  from  the  jugular,  and  this  was  followed 
by  taking  samples  daily  for  about  a  month.  The  horse  was  inocu- 
lated on  January  17,  and  the  blood  which  was  taken  on  January  22 
gave  a  positive  reaction  to  the  complement-fixation  test,  thereby 
proving  that  five  days  after  infection  the  presence  of  glanders  could 
be  ascertained  by  complement  fixation.  The  animal  at  that  time 
showed  no  clinical  indication  of  the  disease,-  but  three  days  later 
(on  the  eighth  day)  the  discharge  from  the  nose  appeared,  and  at 
the  same  time  the  characteristic  swelling  of  the  submaxillary  lymph 
glands  was  noted.  Repeated  tests  made  with  blood  taken  daily  from 
the  horse  gave  always  the  characteristic  reaction.  After  four  weeks 
from  the  time  of  the  infection  the  reaction  appeared  to  be  less  pro- 
nounced, this  probably  being  due  to  the  acuteness  of  the  disease  and 
the  great  amount  of  toxin  which  had  been  produced  continuously  in 
the  animal.  The  testing  of  this  horse  was  not  continued  after  the 
disease  had  affected  the  animal  to  such  an  extent  that  it  appeared 
dangerous  to  continue  the  work,  owing  to  the  extensive  infection 
accompanied  by  a  very  profuse  discharge. 

Since  this  method  of  diagnosis  for  glanders  was  inaugurated  in  this 
laboratory,  large  numbers  of  horses  and  mules  have  been  examined 
in  the  District  of  Columbia,  as  well  as  animals  from  other  parts  of 
the  United  States.  Many  of  the  horses  examined  had  clinical  cases 
of  glanders,  while  others  were  selected  because  they  were  reactors  to 
the  mallein  test,  some  typically,  and  others  atypically.  A  large  pro- 
portion of  the  cases,  however,  were  exposed  or  "  contact "  horses. 
From  the  number  of  tests  already  made — about  1,540 — the  results  indi- 
cate that  in  the  complement  fixation  we  have  a  method  which  in  accu- 
racy is  equal  to  the  tuberculin  test  for  the  diagnosis  of  tuberculosis 
in  cattle.  The  results  of  the  tests  thus  far  conducted  show  that  at 
least  97  per  cent  of  the  cases  of  glanderous  affections  can  be  de- 
termined by  the  complement-fixation  method.  Furthermore,  the 
affected  horses  in  which  a  negative  or  atypical  reaction  occurs  are  as 
a  rule  either  very  old  chronic  cases  of  glanders,  or  those  fresh  cases 
of  infection  tested  during  the  period  of  incubation.  According  to 
Hutyra  and  Marek,^  the  diagnosis  of  glanders  by  the  complement- 
fixation  test  has  already  given  such  accurate  results  that  it  may  be 
considered  as  the  best  method  for  the  determination  of  this  disease 
at  the  present  time.  j 

A  good  opportunity  was  recently  afforded  to  apply  the  test  exten- 
sively in  an  outbreak  of  glanders  which  occurred  during  the  month 
of  January,  1911,  in  a  fashionable  boarding  stable  in  the  District  of 

iSpezielle  Pathologic  und  Theraple  der  Haustlere.     Third  edition,  Band  1,  1910,  p.  717. 


EESULTS  OF  PRACTICAL.  TESTS.  31 

Columbia.  At  this  stable  two  clinical  cases  of  glanders  were  found, 
and  the  physical  examination  of  60  horses  in  the  stable  showed  three 
additional  cases  with  not  typical  but  somewhat  suspicious  symptoms. 
The  testing  of  all  the  horses  in  the  stable  was  immediately  under- 
taken, and  as  a  result  of  the  complement-fixation  tests  14  horses  were 
found  to  be  affected  with  glanders.  Unfortunately  it  was  impossible 
to  obtain  the  consent  of  the  proprietor  to  test  the  horses  with  mallein 
for  a  comparative  study  of  these  two  methods  of  diagnosis. 

Careful  post-mortem  examinations  were  made  on  all  the  reacting 
animals,  and  in  every  instance  lesions  of  glanders  were  found  in  one 
or  more  organs  or  tissues.  In  some  of  the  cases  only  a  few  character- 
istic pulmonary  nodules  were  present,  while  in  others  the  character- 
istic lesions  of  acute  glanders  of  the  lungs  and  liver  were  present. 
There  was  not  a  single  case  among  these  horses  in  which  the  diagnosis 
on  post-mortem  examination  could  have  been  doubted. 

After  removing  and  killing  the  affected  animals  the  stable  was 
thoroughly  cleaned  and  disinfected,  and  a  second  test  was  made  with 
the  blood  of  the  remaining  horses  three  weeks  from  the  time  the  first 
test  had  been  conducted.  On  the  second  test  all  the  horses  except  one 
gave  an  absolutely  negative  reaction.  This  horse  was  destroyed  at 
once,  and  the  post-mortem  examination  revealed  a  caseated  focus  in 
the  left  bronchial  lymph  gland  and  about  twenty  small  nodules  of 
glanders  in  the  lungs,  all  of  which  were  evidently  recent,  having  de- 
veloped without  doubt  during  the  interval  between  the  tests,  as  a 
result  of  the  previous  heavy  exposure. 

In  all  other  cases  where  the  test  has  been  applied  and  in  which 
careful  post-mortem  examinations  were  held  after  the  destruction  of 
the  positive  cases  the  presence  of  glanders  has  been  demonstrated. 
Of  course  not  all  the  animals  which  gave  a  reaction  were  autopsied, 
inasmuch  as  in  some  instances  the  tests  were  made  for  State  officials, 
the  animals  not  being  under  the  jurisdiction  of  the  Department  of 
Agriculture.  Again,  in  other  cases  where  the  animals  were  destroyed, 
no  data  of  the  post-mortem  examinations  were  furnished. 

Among  the  horses  tested  by  the  complement  fixation  there  were 
a  number  of  animals  which  gave  an  atypical  reaction  to  the  mallein 
test,  but  on  the  complement-fixation  test  they  proved  either  absolutely 
positive  or  negative.  Of  these  horses  those  which  gave  a  positive 
reaction  and  were  killed  proved  to  be  glandered. 

Table  5  shows  the  comparative  results  obtained  with  the  mallein 
and  complement- fixation  tests: 


82 


DIAGNOSIS   OF   GLANDERS. 


Table  5. — Comparative  results  with  maJlein 

and 

complemewt-fixation  tests. 

Positive  to  malleln  test. 

Negative  to  mal- 
lein  test. 

Atypical  reaction  to  mallein 

test. 

Locality. 

To- 
tal. 

Response 
to  com- 
plement- 
fixation 

test. 

Post-mor- 
tem. 

To- 
tal. 

Response 
to  com- 
plement- 
fixation 
test. 

To- 
tal. 

Response 
to  com- 
plement- 
fixation 
test. 

Post-mor- 
tem. 

Total 
ani- 
mals. 

Posi- 
tive. 

Nega- 
tive. 

Posi- 
tive. 

Nega- 
tive. 

Posi- 
tive. 

Nega- 
tive. 

Posi- 
tive. 

Nega- 
tive. 

Posi- 
tive. 

Nega- 
tive. 

Connecticut 

9 
9 
6 
6 
7 
18 
1 

35 
19 
1 
6 
5 
2 
6 
6 
2 
1 

8 
6 
4 
3 

"iY 

1 

26 

19 

...... 

6 
2 
4 
2 
2 

1 
4 
2 
2 

7 

1 

3 

1 
"■■■4' 

1 

4 
6 

...... 

....„ 

1 

10 

Florida 

9 

8 

1 

6 
4 
3 

12 
6 
9 
8 

12 
1 
6 
2 
1 

24 

Illinois 

10 

Indiana 

11 
32 
8 

2 

9 
32 
3 

2 
12 
6 
6 
4 
6 
1 
3 
2 

1 

'"'h' 

19 

Maine 

7 

61 

Michigan 

27 

Missouri 

10 

Montana 

9 

17 
11 

4 
11 

13 

60 

Nortli  Dakota . . . 

42 

Pennsylvania . . . 

1 

1 

2 

Nebraska 

12 

Wvoming 

7 

Washington 

3 

California 

2 
8 

6 

Texas 

4 
1 

...... 

4 

9 

Oregon 

3 

Canada 

1 

1 

Miscellaneous . . . 

29 

20 

9 

20 

29 

Total 

137 

103 

34 

3 

7 

83 

25 

58 

104 

44  1      61 

22  1       8 

325 

In  addition  to  the  above  there  have  been  1,217  tests  made  upon 
horses  with  the  complement- fixation  method  alone,  and  we  have  also 
tested  by  this  method  the  blood  of  one  lion,  which  gave  a  positive 
reaction,  and  the  blood  of  one  human  suspected  of  having  glanders, 
which  proved  negative.    These  two  results  were  later  substantiated. 

Of  the  above-mentioned  1,217  tests,  643  were  conducted  on  horses 
at  Washington,  D.  C,  and  of  these  21  gave  a  positive  reaction,  all  of 
which  were  subsequently  confirmed  on  post-mortem.  The  remaining 
575  were  from  miscellaneous  sources,  78  of  which  were  positive,  while 
497  were  negative. 

In  the  323  cases  in  Table  5  wherein  the  two  tests  are  compared, 
the  mallein  test  was  confirmed  by  the  complement-fixation  test  in  161 
cases  and  was  not  confirmed  in  59  cases.  There  were  104  atypical 
reactions  to  mallein  which  were  definitely  diagnosed  by  complement 
fixation — 44  positive  and  60  negative.  Seven  of  the  Maine  reactors  to 
mallein  and  three  atypical  reactors  have  been  examined  post-mortem 
without  showing  any  evidence  of  glanders. 

In  order  to  determine  whether  the  fixation  of  the  complement  may 
be  obtained  occasionally  in  normal  horses  or  in  horses  affected  with 
various  diseases  other  than  glanders,  a  number  of  tests  were  made 
with  the  blood  of  apparently  normal  horses  and,  also,  with  horses 
suffering  with  various  infectious  and  noninfectious  diseases.  One  of 
these  tests  was  made  with  the  blood  of  a  horse  affected  with  swamp 
fever,  in  which  the  temperature  registered  106.2°  F. ;  other  tests  were 
made  with  blood  from  horses  affected  with  distemper,  dourine,  influ- 
enza, pneumonia,  heaves,  lameness,  fistulous  withers,  forage  poison- 
ing, etc. ;  but  in  all  these  cases  negative  reactions  were  obtained. 

From  these  results  the  specificity  of  the  test  can  be  readily  appre- 
ciated. Animals  affected  with  glanders  will  give  a  positive  reaction. 
Normal  animals  or  those  affected  with  diseases  other  than  glanders 
will  give  no  reaction. 


UC  SOUTHERN  REGIONAL  LIBRARY  FACILITY 


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